Methods of using dyes in association with nucleic acid staining or detection and associated technology

ABSTRACT

Methods of using dyes and associated technology are provided. A dye, such as a monomeric dye or a dimeric dye, may be used in a nucleic acid gel staining application and/or a nucleic acid detection application. Such a dye and a salt that comprises an anion that is associated with a strong acid and a cation that is associated with a strong base may be used in such an application. A dimeric dye, such as a dimeric dye capable of forming a hairpin-like structure, may be used to stain and/or detect nucleic acids via a release-on-demand mechanism. A dimeric dye having low background fluorescence in the absence of nucleic acids and high fluorescence in the presence of nucleic acids, upon binding therewith, may be used to stain and/or detect nucleic acids.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional application of U.S. application Ser.No. 12/484,968, filed on Jun. 15, 2009, which is a continuation of U.S.application Ser. No. 11/377,254, filed on Mar. 16, 2006, issued as U.S.Pat. No. 7,601,498 on Oct. 13, 2009, which claims benefit under 35U.S.C. §119(e) to U.S. Provisional Application No. 60/663,613 filed onMar. 17, 2005, each of which is hereby incorporated by reference in itsentirety.

BACKGROUND

Fluorescent dyes or stains can be used in the detection of nucleicacids, such as DNA and RNA, and biological samples involving nucleicacids. Nucleic acid polymers, such as DNA and RNA, are involved in thetransmission of genetic information from one generation to the next andthe routine functioning of living organisms. Nucleic acids are thus ofinterest and the objects of study. Fluorescent nucleic acid dyes thatspecifically bind to nucleic acids and form highly fluorescent complexesare useful tools for such study. These dyes can be used to detect thepresence and quantities of DNA and RNA in a variety of media, includingpure solutions, cell extracts, electrophoretic gels, micro-array chips,live or fixed cells, dead cells, and environmental samples.

Nucleic acids may be separated via gel electrophoresis, wherein thenucleic acids are placed in a gel, such as an agarose gel or apolyacrylamide gel, and electrophoretically separated. The separatednucleic acids may then be visualized. According to one method, referredto as post-gel staining or post-staining, the gel may be stained with anucleic acid dye solution and then viewed with an appropriatetransilluminator. According to another method, referred to as pre-caststaining, the gel may be premixed with the dye during gel preparation,prior to visualization. Such a gel that is premixed, or pre-embedded,with a nucleic acid dye may be referred to as a pre-cast gel. Nucleicacids separated by a pre-cast gel can be visualized directly with atransilluminator.

Several dyes have been used as nucleic acid gel stains. For example,ethidium bromide (EB), a relatively inexpensive and adequately sensitivedye, has been used as a nucleic acid gel stain. EB is associated withseveral disadvantages however. First, EB is known to be a powerfulmutagen and carcinogen, requiring special handling and waste disposalprocedures (M. J. Waring, J. Mol. Biol. I. 13, 269 (1965); McCann etal., Proc. Natl. Acad. Sci. USA, 72, 5135 (1975); and Fukunaga et al.,Mutation Res. 127, 31 (1984)). Second, EB has significant intrinsicfluorescence, which contributes to background fluorescence, particularlyfor post-gel staining. This intrinsic fluorescence is significant in thesense that actual DNA bands, particularly, any relatively weak bands,may be indistinguishable relative to the background. Consequently,post-gel staining with EB typically requires a destaining step to removebackground fluorescence. The extra destaining step results in not onlyinconvenience, but also in additional human exposure to the toxicmaterial. Third, when EB is used in pre-cast gel staining, the dye tendsto migrate in a direction opposite the direction of DNA migration. Thisusually leaves one end of the gel with a high dye concentration, whichcontributes to high background fluorescence, and the other end of thegel with an insufficient dye concentration, which lowers detectionsensitivity.

Asymmetric cyanine dyes have been developed as alternatives to EB fornucleic acid gel stain applications. These dyes have been reported to bemore sensitive than EB and to be more efficiently excited by the 488 nmargon laser. The asymmetric cyanine dye, SYBR Green I, has been marketedas both a pre-cast gel stain and a post-gel stain. However, the SYBRGreen I dye has only limited stability in commonly used electrophoresisbuffers, such that pre-cast gels prepared with the dye have to be usedwell within 24 hours before losing utility. The asymmetric cyanine dye,SYBR Gold, has been described as being more sensitive than SYBR Green Ias a post-gel stain. However, the SYBR Gold dye cannot be used as apre-cast gel stain because of its low stability. Another asymmetriccyanine dye, SYBR Safe, has been developed as an alternative to SYBRGreen I and EB due to its low mutagenicity (U.S. Patent ApplicationPublication No. 2005/0239096). However, this alternative dye is lesssensitive than desired.

Development of fluorescent dyes or the making or the use thereof isdesirable.

SUMMARY OF THE INVENTION

A method of producing, designing or using a fluorescent dye suitable foruseful application, such as in nucleic acid gel staining, for example,is provided. The method may involve covalently linking two monomericdyes via a bridge that is flexible and substantially neutral (forexample, neutral or slightly charged).

A fluorescent dye suitable for useful application, such as thatdescribed above, for example, is provided. A dimeric dye, which may beproduced according to a method described herein, may form a hairpinstructure, which, it is believed, enables the dye to stain immobilizednucleic acids, such nucleic acids immobilized in a gel matrix, via arelease-on-demand mechanism, as further described herein. A dye may haveat least one feature, or all of the following features: relatively low“fluorescence background” (fluorescence in the absence of nucleicacids), if any, and ideally, no fluorescence background; relatively lowtoxicity, and ideally, no toxicity; relatively high fluorescent signalstrength; and relative high stability. The dye is preferably better asto at least one of these features, and more preferably, as to all ofthese features, than an existing dye, such as EB, SYBR Green I or SYBRSafe, merely by way of example.

Dimeric nucleic acid dyes or stains that are capable of intramoleculardimer formation, or the formation of a hairpin structure, are provided.It is believed that a hairpin-shaped dye is non-fluorescent or isminimally fluorescent by itself, but becomes highly fluorescent in thepresence of nucleic acids. It is believed that nucleic acid binding ofthe dye occurs via an intermediate state wherein the dye forms, in part,an open random conformation. It is further believed that this openrandom conformation of the dye exists in a small quantity and inequilibrium with the hairpin state. It is believed that as the amount ofnucleic acids increases, an equilibrium shift from the hairpin statetoward the nucleic acid-bound state of the dye occurs, such that thestrength of the resulting fluorescence signal is substantially linearlyproportional to the amount of nucleic acids present.

The above-described mechanism, which may be referred to as arelease-on-demand mechanism of DNA staining, may be desirable forvarious applications, such as nucleic acid gel staining, for example.Merely by way of explanation, it is believed that formation of thehairpin structure renders the “effective dye concentration” low, suchthat a dye generally has low background fluorescence and low toxicity.Thus, as compared with previous dyes, such as EB or SYBR Green I, forexample, a higher concentration of a dye described herein may be used innucleic acid gel staining. This higher concentration of dye may increaseDNA detection sensitivity, perhaps significantly, such as up to tenfoldgreater relative to that associated with EB, for example.

A method of determining presence or absence of nucleic acid in a sampleis provided. When the sample is exposed to a matrix or a surface,nucleic acid present in the sample, if any, may become immobilizedrelative to the matrix or the surface. The method comprises exposing thesample to a fluorescent nucleic acid dye having the formula:

wherein BRIDGE is a substantially aliphatic, substantially neutrallinker comprising from about 8 to about 150 non-hydrogen atoms,inclusive; Q₁ is a fluorescent nucleic acid dye constituent; Q₂ is afluorescent nucleic acid dye constituent; and Q₁ and Q₂ may be the sameor different. This exposure is such that if nucleic acid is present inthe sample, a complex of the fluorescent nucleic acid dye and thenucleic acid is formed. The method comprises detecting fluorescenceassociated with the complex, or a lack thereof. The method may beassociated with pre-cast nucleic acid gel staining or post-nucleic acidgel staining, merely by way of example.

A kit for determining presence or absence of nucleic acid in a sample isprovided. The kit comprises the fluorescent nucleic acid dye justdescribed, and information concerning use of the fluorescent nucleicacid dye. The kit may comprise, optionally, a buffer and/or a gelmatrix, at least one material for forming a gel matrix, a surface, or atleast one material for forming a surface. The fluorescent nucleic aciddye may be in an aqueous solution or in a gel matrix, such as a agarosegel matrix, for example.

A method of post-nucleic acid gel staining a sample is provided. Whenthe sample is exposed to a matrix or a surface, nucleic acid present inthe sample, if any, may become immobilized relative to the matrix or thesurface. The method comprises providing an aqueous solution comprising afluorescent nucleic acid dye and a salt, and exposing the sample to theaqueous solution. The salt comprises an anion that would be sufficientas a component of a strong acid and a cation that would be sufficient asa component of such a strong acid a strong base.

These and various other aspects, features, and embodiments are furtherdescribed herein.

BRIEF DESCRIPTION OF THE DRAWINGS

A detailed description of various aspects, features and embodiments isprovided herein with reference to the accompanying drawings, which arebriefly described below. The drawings are illustrative and are notnecessarily drawn to scale. The drawings illustrate various aspects orfeatures and may illustrate one or more embodiment(s) or example(s) inwhole or in part. A reference numeral, letter, and/or symbol that isused in one drawing to refer to a particular element or feature may beused in another drawing to refer to a like element or feature.

FIG. 1 (FIG. 1) is a schematic illustration of DNA binding via arelease-on-demand mechanism, in which three conformation states of thedye are in substantial equilibrium.

FIG. 2 (FIG. 2) is a graphical representation of normalized absorbanceversus wavelength (nm), or normalized absorption spectra, of a) adimeric dye, AOAO-7 (∘), and b) a monomeric AO dye, DMAO (Δ), in PBSbuffer.

FIG. 3 (FIG. 3) is a graphical representation of normalized absorbanceversus wavelength (nm), or normalized absorption spectra, of a) adimeric dye, AOAO-7 (●), and b) a monomeric AO dye, DMAO (▴), in abuffer and in the presence of DNA.

FIG. 4 (FIG. 4) is a graphical representation of relative fluorescenceversus wavelength (nm), or fluorescence emission spectra, of DMAO (Δ)and AOAO-7 (∘) in PBS buffer before DNA addition, a) and c),respectively, and DMAO (▴) and AOAO-7 (●) in PBS buffer after DNAaddition, b) and d), respectively.

FIG. 5 (FIG. 5) is a graphical representation of normalized absorbanceversus wavelength (nm), or normalized absorption spectra, of a) TOTO-1(prepared according to U.S. Pat. No. 5,582,977) (darker line), and b)TOTO-3 (lighter line), in a buffer.

FIG. 6 (FIG. 6) is a graphical representation of normalized absorbanceversus wavelength (nm), or normalized absorption spectra, of a) TOTO-1according to U.S. Pat. No. 5,582,977) (darker line), and b) TOTO-3(lighter line), in a buffer and in the presence of DNA.

FIG. 7 (FIG. 7) includes a graphical representation of relativefluorescence versus DNA concentration (μg/mL), or a titration, ofsingle-stranded DNA (⋄), and double-stranded DNA (♦), in solution and inthe presence of AOAO-12 (at 0.2 μM). FIG. 7 also includes an insetgraphical representation of relative fluorescence versus DNAconcentration that shows a substantially linear relationship between thetwo.

FIG. 8 (FIG. 8) includes photograph A of a gel upon post-DNA gelstaining with SYBR Safe at 1× in TBE; photograph B of a gel uponpost-DNA gel staining with SYBR Green I at 1× in TBE; and photograph Cof a gel upon post-DNA gel staining with Dye No. 20 of Table 1 at 3.6 μMin TBE, each as viewed via a 254-nm UV light transilluminator. Theamount of DNA loading per lane, or column, from left to right, as shownin each of the photographs, was 200 ng, 100 ng, 50, ng, and 25 ng,respectively. Photographs A, B and C were taken using a SYBR filter andPolaroid 667 black-and-white print film.

FIG. 9 (FIG. 9) includes photograph A of a gel upon post-DNA gelstaining with SYBR Safe at 1× in TBE; photograph B of a gel uponpost-DNA gel staining with SYBR Green I at 1× in TBE; and photograph Cof a gel upon post-DNA gel staining with Dye No. 29 (TOTO-13) of Table 1at 3.6 μM in water with 0.1 M NaCl, each as viewed via a 254-nm UV lighttransilluminator. The amount of DNA loading per lane, or column, fromleft to right, as shown in each of the photographs, was 200 ng, 100 ng,50, ng, and 25 ng, respectively. Photographs A, B and C were taken usinga SYBR filter and Polaroid 667 black-and-white print film.

FIG. 10 (FIG. 10) is a graphical representation of the relativefluorescence intensity of excitation and emission spectra (nm) of DyeNo. 29 (TOTO-13) of Table 1 in the presence of dsDNA.

FIG. 11 (FIG. 11) is a photograph of a gel upon post-DNA gel stainingwith Dye No. 29 of Table 1 at 3.6 3.6 μM in water with 0.1 M NaCl, asviewed via a Dark Reader visible light transilluminator from ClareChemical Research (Dolores, Colo. (CO)). The amount of DNA loading perlane, or column, from left to right, as shown in the photograph, was 200ng, 100 ng, 50, ng, and 25 ng, respectively. The photograph was takenusing a SYBR filter and Polaroid 667 black-and-white print film.

FIG. 12 (FIG. 12) includes a photograph of a gel upon pre-cast DNA gelstaining with Dye No. 35 (ET-27) of Table 1 at 1.2 μM in TBE and aphotograph of a gel upon pre-cast DNA gel staining with EB at 1.3 μM inTBE, each as viewed via a 300-nm UV light transilluminator. The amountof DNA loading per lane, or column, from left to right, as shown in eachof the photographs, was 200 ng, 100 ng, 50, ng, and 25 ng, respectively.The photographs were taken using a SYBR filter and Polaroid 667black-and-white print film.

FIG. 13 (FIG. 13) is a graphical representation of normalized absorbanceof Dye No. 35 (ET-27) of Table 1 at about 1.2 μM in TBE and normalizedabsorbance of SYBR Gold at 1× effective working concentration in TBE,over time (hours) at room temperature.

FIG. 14 (FIG. 14) includes a photograph A of a gel upon post-DNA gelstaining with SYBR Green I at 1× in 1×TBE; a photograph B of a gel uponpost-DNA gel staining with SYBR Green I at 1× in 1×TBE with 0.1 M NaCl;a photograph C of a gel upon post-DNA gel staining with SYBR Green I at1× in H₂O with 0.1 M NaCl; a photograph D of a gel upon post-DNA gelstaining with Dye No. 29 of Table 1 at 3.6 μM in 1×TBE; a photograph Eof a gel upon post-DNA gel staining with Dye No. 29 of Table 1 at 3.6 μMin 1×TBE with 0.1 M NaCl; and a photograph F of a gel upon post-DNA gelstaining with Dye No. 29 of Table 1 at 3.6 μM in H₂O with 0.1 M NaCl,each as viewed via a 254-nm UV light transilluminator. The amount of DNAloading per lane, or column, from left to right, as shown in each of thephotographs, was 200 ng, 100 ng, 50, ng, and 25 ng, respectively. Thephotographs were taken using a SYBR filter and Polaroid 667black-and-white print film.

DESCRIPTION

Fluorescent dyes or stains may be useful in various applications, suchas nucleic acid detection, for example. Methods associated withfluorescent dyes or stains, such as methods of use thereof, for example,may also be useful. Dimeric nucleic acid dyes, such as those having lowbackground fluorescence in the absence of nucleic acids and relativelygreater fluorescence in the presence of nucleic acids, such asimmobilized nucleic acids, for example, may also be useful. Dimericnucleic acid dyes may be useful in various applications, such as nucleicacid detection in gels, for example. Useful dyes or stains, andassociated technology, such as methods of using same, for example, aredescribed herein.

Herein, it will be understood that a word appearing in the singularencompasses its plural counterpart, and a word appearing in the pluralencompasses its singular counterpart, unless implicitly or explicitlyunderstood or stated otherwise. Further, it will be understood that forany given component described herein, any of the possible candidates oralternatives listed for that component, may generally be usedindividually or in any combination with one another, unless implicitlyor explicitly understood or stated otherwise. Additionally, it will beunderstood that any list of such candidates or alternatives, is merelyillustrative, not limiting, unless implicitly or explicitly understoodor stated otherwise. Still further, it will be understood that anyfigure or number or amount presented herein is approximate, and that anynumerical range includes the minimum number and the maximum numberdefining the range, whether or not the term “inclusive” or the likeappears, unless implicitly or explicitly understood or stated otherwise.Additionally, it will be understood that any permissive, open, oropen-ended language encompasses any relatively permissive to restrictivelanguage, open to closed language, or open-ended to closed-endedlanguage, respectively, unless implicitly or explicitly understood orstated otherwise. Merely by way of example, the word “comprising” mayencompass “comprising”13 , “consisting essentially of”—, and/or“consisting of”—type language.

Various terms are generally described or used herein to facilitateunderstanding. It will be understood that a corresponding generaldescription or use of these various terms applies to correspondinglinguistic or grammatical variations or forms of these various terms. Itwill also be understood that a general description or use or acorresponding general description or use of any term herein may notapply or may not fully apply when the term is used in a non-general ormore specific manner. It will also be understood that the terminologyused or the description provided herein, such as in relation to variousembodiments, for example, is not limiting. It will further be understoodthat embodiments described herein or applications described herein, arenot limiting, as such may vary.

Generally, the terms “stain” and “dye” may be used interchangeably andrefer to an aromatic molecule capable of absorbing light in the spectralrange of from about 250 nm to about 1,200 nm, inclusive. Generally, theterm “dye” may refer to a fluorescent dye, a non-fluorescent dye, orboth. Generally, the term “fluorescent dye” refers to a dye capable ofemitting light when excited by another light of appropriate wavelength.

Generally, the term “fluorescence quencher” refers to a molecule capableof quenching the fluorescence of another fluorescent molecule.Fluorescence quenching can occur via at least one of the three ways. Thefirst type of fluorescence quenching occurs via fluorescence resonanceenergy transfer (FRET) (Förster, Ann. Phys. (1948); and Stryer, et al.,Proc. Natl. Acad. Sci. (1967)), wherein a quencher absorbs the emissionlight from a fluorescent molecule. The absorption peak of a FRETquencher usually has to have significant overlap with the emission peakof a fluorescent dye for the FRET quencher to be an efficientfluorescent quencher. A FRET quencher is typically a non-fluorescentdye, but can also be a fluorescent dye. When a quencher is a fluorescentdye, only the absorption property of the dye is utilized. A second typeof fluorescence quenching occurs via photo-induced electron transfer(PET), wherein the quencher is an electron-rich molecule that quenchesthe fluorescence of a fluorescent molecule by transferring an electronto the electronically excited dye. A third type of fluorescencequenching occurs via dye aggregation, such as H-dimer formation, whereintwo or more dye molecules are in physical contact with one another,thereby dissipating the electronic energy into the vibrational modes ofthe molecules. This type of contact fluorescence quenching can occurbetween two identical fluorescent dyes, or between two differentfluorescent dyes, or between a fluorescent dye and a FRET quencher, orbetween a fluorescent dye and a PET quencher. Other types offluorescence quenchers, though not used as commonly, include stable freeradical compounds and certain heavy metal complexes.

Generally, the term “nucleic acid” refers to double-stranded DNA(dsDNA), single-stranded DNA (ssDNA), double-stranded RNA (dsRNA),single-stranded RNA (ssRNA), and/or derivatives thereof. A nucleic acidmay be natural or synthetic.

Generally, the term “fluorescent nucleic acid stain” or “fluorescentnucleic acid dye” refers to a dye capable of binding to a nucleic acidto form a fluorescent dye-nucleic acid complex. A fluorescent nucleicacid dye is typically non-fluorescent or weakly fluorescent by itself,but becomes highly fluorescent upon nucleic acid binding. Generally, theterm “non-fluorescent, nucleic acid-binding molecule” refers to anucleic acid-binding molecule that may or may not be a dye and that doesnot become fluorescent upon binding to nucleic acid. Generally, the term“fluorescent DNA dye” refers to a dye that becomes fluorescent uponbinding to DNA. Generally, the term “fluorescent, non-nucleic acid dye”refers to a fluorescent dye that does not bind to nucleic acid.Generally, the term “non-fluorescent, non-nucleic acid dye” refers to adye that is neither fluorescent nor nucleic acid-binding. Such a dye iscommonly called a fluorescence quencher. Frequently, a fluorescencequencher is used to form a FRET pair with a fluorescent dye. Generally,the term “reporter dye” refers to a fluorescent dye whose emittedfluorescence contributes to the final detected fluorescence signal.

Generally, the term “TBE” refers to an aqueous buffer comprising about89 mM Tris, about 89 mm borate, and about 2 mM EDTA, with a pH of about8.3; the term “TAE” refers to an aqueous buffer comprising about 40 mMTris, about 20 mM acetate, and about 2 mM EDTA, with a pH of about 8.1;and the term “EB” refers to ethidium bromide.

Generally, a salt that comprises a cation that is associated with astrong base and an anion that is associated with a strong acid refers toa salt that comprises such a cation and such an anion from whateversource, whether from the strong acid or strong base or from any othersuitable source. The strong base may have a pKa of about 10 or greater,and the strong acid may have a pKa of about 2 or less. In this regard,“a cation that is associated with a strong base” generally refers to acation that would be sufficient as a component of such a strong base,but need not actually be such a component, and “an anion that isassociated with a strong acid” generally refers to an anion that wouldbe sufficient as a component of such a strong acid, but need notactually be such a component. Merely by way of example, the salt may beone that when dissolved in water is sufficiently ionized, such as on theorder of at least 90% ionized, for example. A concentration of such asalt in solution may be from about 5 mM to about 0.5 M, inclusive, suchas about 0.05 M to about 0.2 M, or about 0.1 M, for example. Such a saltmay be non-buffering. Generally, when a non-buffering salt is dissolvedin water, it fully dissociates into the cation and anion withoutsignificantly changing the pH of the water. In this regard, asignificant change may be a pH change of ±0.5, inclusive, such as ±0.3,inclusive, for example. Examples of such salts include, but are notlimited to, sodium chloride, potassium chloride, sodium sulfate,potassium sulfate, sodium bromide, potassium bromide,tetramethylammonium chloride, magnesium chloride, and/or the like.

In general, fluorescent nucleic acid dyes can be classified into twomajor classes: intercalators and minor groove-binders. Generally,fluorescent intercalators are dyes that bind to double-stranded DNA(dsDNA) or double-stranded RNA (dsRNA) by inserting themselves inbetween a neighboring base pair. Generally, minor groove-binders aredyes that bind to the minor groove of double-stranded DNA. There arestill other dyes that may bind to nucleic acids via multiple modes,including electrostatic interaction between a positively charged dye andthe negatively charged nucleic acid.

A method for designing a fluorescent nucleic acid dye for detectingimmobilized nucleic acids, such as for detecting nucleic acids in gels,is now described. The method comprises covalently linking two monomericdyes with a suitable linker to form a dimeric dye. The dimeric dye, whenin solution, assumes a predominantly hairpin-like conformation due tointramolecular dimer formation. This hairpin-like conformation or stateof the dye is inactive with respect to nucleic acids, or incapable ofinteracting or groove-binding with nucleic acids. It is believed thatthe dye, when in solution and in the presence of nucleic acids, alsoassumes an open random conformation or state, which exists in smallquantity and in substantial equilibrium with the hairpin conformation.The open random conformation or state of the dye is active with respectto nucleic acids, or capable of interacting or binding with nucleicacids. It is believed that when the dye is in the presence of anincreasing amount of nucleic acids, an equilibrium shift from thehairpin state toward the intermediate, open random state, or DNA-bindingstate, occurs. It is believed that this mechanism, sometimes referred toas a “release-on-demand DNA-binding mechanism,” reduces backgroundfluorescence and sometimes may also reduce the toxicity of the dye. As aconsequence, the dye may be used in nucleic acid gel staining at ahigher concentration than might otherwise be possible, and thus, mayprovide for greater nucleic acid detection sensitivity than mightotherwise be possible.

The dimeric dye may posses any number of desirable characteristics. Byway of example, such a dye may have a background fluorescence that isreduced relative to that of its monomeric dye constituents. Relativelylow background fluorescence generally corresponds to relatively enhancednucleic acid detection sensitivity. Thus, such a dimeric dye isgenerally associated with enhanced nucleic acid detection sensitivity.Further by way of example, a dimeric dye may be more thermally and/orhydrolytically stable than SYBR Green I. Still further by way ofexample, a dimeric dye may be less toxic, particularly less mutagenic,than some of the dyes previously used in nucleic acid gel stains.

A fluorescent dimeric nucleic acid dye may have the general structure(Structure 1) set forth directly below.

In relation to the brief summary and the description, references to adimeric dye are to a dimeric dye of Structure 1. In Structure 1, each ofQ₁ and Q₂ is a fluorescent nucleic acid dye. Q₁ and Q₂ may be selectedand combined in a manner to encourage or to ensure desired properties ofthe resulting dimeric dye. BRIDGE may be positively charged to arelatively limited extent or substantially neutral in charge, and may bea substantially flexible constituent that facilitates intramoleculardimer formation to produce the dimeric dye.

BRIDGE may be a substantially flexible linker molecule, having no morethan one positive charge. BRIDGE may be a substantially neutral andsubstantially flexible linker molecule. The constituents of BRIDGE maybe selected to provide such limited positive charge or substantialneutrality. The property of substantial neutrality, which includesactual neutrality, is discussed further below. The property ofsubstantial flexibility is generally related to the substantiallyaliphatic nature, which includes actual aliphatic nature, of BRIDGE.This substantial aliphatic nature generally refers to thenon-aromaticity of BRIDGE, or non-rigidity of BRIDGE.

In Structure 1, BRIDGE is covalently attached to Q₁ and Q₂. In a dimericdye, BRIDGE may generally have from about 8 to about 150 non-hydrogenatoms, inclusive; from about 10 to about 100 non-hydrogen atoms,inclusive; from about 15 to about 80 non-hydrogen atoms, inclusive; orfrom about 20 to about 50 non-hydrogen atoms, inclusive.

BRIDGE may incorporate at least one independentnucleic-acid-binding-enhancing-group (NABEG). A NABEG is a moietycapable of binding to nucleic acids in the form of electrostatic,hydrophobic, or hydrogen-bonding interactions. Merely by way of example,a NABEG may be selected from primary amines; secondary amines; tertiaryamines; ammoniums; amidines; aryl groups, optionally comprising heteroatoms selected from N, O, S, and any combination thereof; moietieshaving bonds comprising hetero atoms of high electronegativity; and anycombination thereof.

Primary, secondary and tertiary amines and amidines are basic groups andtherefore are positively charged or at least partially positivelycharged at physiological pH. Ammonium groups, or quaternized nitrogengroups, are permanently positively charged. Generally speaking,positively charged or partially positively charged groups enhance thenucleic acid binding of the dye via electrostatic interaction, aproperty that may be exploited in the development of highly sensitivefluorescent nucleic acid stains. It is generally undesirable to useBRIDGE having excessive positive charges to produce a dimeric dye. Asuitable BRIDGE of a dimeric dye may comprise no more than one positivecharge. BRIDGE may be a substantially flexible and neutral orsubstantially neutral linker. In this context, substantially neutralityrefers to slight charge. By way of example, BRIDGE could comprise aweakly basic constituent, such as a pyridine group or a pyrazine group,for example, such that when it is in aqueous solution, a very smallamount of positive charges may be present. Further by way of example, ina case in which BRIDGE comprises at least one neutral NABEG, the exactamount of positive charge is generally related to the pK_(a) of theNABEG. Generally, the higher the pK_(a) of the NABEG, the more likelythe NABEG is protonated and thus, positively charged. By way of example,a suitable weakly basic NABEG group may have a pK_(a) of about 11 orless, inclusive; about 8 or less, inclusive; or about 7 or less,inclusive.

There may be a tendency to form an intramolecular dimer, primarilyH-dimer, which may be a particularly useful property in the nucleic aciddye produced. For example, in the case of a dimeric dye, H-dimerformation produces a hairpin-like structure, wherein H-dimer forms astem portion of the hairpin and BRIDGE forms a curved portion, asschematically illustrated in FIG. 1. The phenomenon of H-dimer formationin connection with certain dyes has been described in West, et al., J.Phys. Chem. (1965); Rohatgi, et al., J. Phys. Chem. (1966); Rohatgi, etal., Chem. Phys. Lett. (1971); and Khairutdinov, et al., J. Phys. Chem.(1997). Formation of an intramolecular H-dimer may be facilitated whenBRIDGE is a flexible and neutral or substantially neutral hydrocarbonlinker, optionally comprising one or more neutral NABEG(s).

H-dimer formation may be characterized by a large blue shift of the dyeabsorption spectrum. By way of example, the absorption spectra of amonomeric dye AO (acridine orange) and a related dimeric dye, AOAO-7,that forms an intramolecular dimer, are shown in FIG. 2. The 471 nm peakassociated with the AOAO-7 dimer indicates intramolecular H-dimerformation. The absorption spectra of both the monomer and the dimerbecome similar once DNA-binding occurs, indicating the opening up of thehairpin structure. By way of example, as shown in FIG. 3, thedisappearance of the 471 nm peak from AOAO-7 dimer indicates the openingup of the hairpin structure upon DNA binding.

H-dimer formation in a dimeric dye may be associated with two majorbenefits. One of the major benefits is a reduction, sometimes dramatic,in background fluorescence, coupled with a substantial increase influorescence upon DNA-binding, as demonstrated by a large gain in thefluorescence signal. This benefit may be appreciated by comparing thefluorescence spectra of a monomeric acridine orange dye, DMAO, and adimeric acridine orange dye, AOAO-7, in the absence and presence of DNA.For example, as shown in FIG. 4, relative to the monomeric DMAO dye, thedimeric AOAO-7 dye is associated with lower background fluorescence andhigher fluorescence upon binding to DNA.

Intramolecular dimer-associated fluorescence quenching may be soefficient that a dimeric dye may be constructed from at least onemonomeric dye that is not normally considered to be very desirable, suchas at least one monomeric dye that has high background fluorescence, forexample. An example of this is shown in FIG. 4, which features acridineorange (AO) and a dimer thereof. Although AO is one of the earliestknown nucleic acid-binding dyes and has desirable wavelengths, it hasnot been widely used for nucleic acid detection because of itsrelatively high background fluorescence. As demonstrated in FIG. 4,relative to the monomeric AO dye, the dimeric dye AOAO-7 has much lowerbackground fluorescence.

H-dimer formation in a dimeric dye may be associated with another majorbenefit. This unexpected benefit is that H-dimer formation in a dimericdye may significantly reduce the toxicity, particularly mutagenicity, ofthe dye. In this regard, a significant reduction in mutagenicity may beon the order of at least about 20% relative to EB, as measured using theAmes Test or an equivalent test. It is believed that reducedmutagenicity may be at least partly attributable to reductions in thecell membrane-permeability and the effective concentration of the dye.The molecular weight of a dimeric dye is generally substantially orsignificantly larger, such as about two times larger, for example, thanthe molecular weights of known nucleic acid gel stains. Generally, amolecule having a larger molecular weight has more difficultypenetrating cell membranes than a molecule having a smaller molecularweight. As such, a molecule having a large molecular weight may berelatively less likely to enter a cell and cause cell damage. As to adimeric dye molecule that successfully enters a cell, the effectiveconcentration of the dye associated with the molecule is generallyrelatively small because of H-dimer formation. As such, the dimeric dyemolecule may be relatively less likely to cause cell damage once itenters a cell.

BRIDGE may have the formula (Formula 1) set forth directly below.-L-[A¹-(CH₂)_(α)-]_(a)[A²-(CH₂)_(β)-]_(b)[A³-(CH₂)_(γ)-]_(c)[A⁴-(CH₂)_(δ)-]_(d)[A⁵-(CH₂)_(ε)-]_(e)[A⁶—(CH₂)_(ζ)-]_(f)[⁷-(CH₂)_(η)-]_(g)[A⁸-(CH₂)_(θ)-]_(h)[A⁹-(CH₂)_(ι)-]_(i)-A¹⁰-L-  Formula1In Formula 1, each L is part of BRIDGE and is covalently linked to Q₁ orQ₂. Each L is independently a moiety comprising a single bond; apolymethylene unit having 1 carbon to about 12 carbons, inclusive,optionally comprising at least one hetero atom selected from N, O and S;or an aryl group optionally comprising at least one hetero atom selectedfrom N, O and S. The subscripts associated with the (CH₂) methyleneunits, namely, α, β, γ, δ, ε, ζ, η, θ, and ι, may be the same ordifferent, each independently indicating the size of the associatedmethylene unit and, independently, being zero or an integer from 1 toabout 20, inclusive, or from 1 to about 12, inclusive. The subscriptsassociated with the bracketed portions of Formula 1, namely, a, b, c, d,e, f, g, h, and i, may be the same or different, each independentlyindicating the size of the associated bracketed portion of the formulaand, independently, being zero or an integer from 1 to about 20,inclusive, or from 1 to about 10, inclusive, or from 1 to about 5,inclusive.

A¹, A², A³, A⁴, A⁵, A⁶, A⁷, A⁸, A⁹, and A¹⁰ may be the same ordifferent, each independently, being anucleic-acid-binding-enhancing-group (NABEG); a branched alkyloptionally comprising at least one hetero atom selected from N, O and S;or at least one saturated 5- or 6-membered ring optionally comprising atleast one hetero atom selected from N, O and S. A¹, A², A³, A⁴, A⁵, A⁶,A⁷, A⁸, A⁹, and A¹⁰ may be such that BRIDGE comprises at most onepositive charge, or is substantially neutral, and in the latter case,each of these constituents, independently, may itself be substantiallyneutral, which includes actual neutrality. NABEGs may be selected frommoieties comprising at least one bond linkage that comprises at leastone hetero atom of high electronegativity or S; and aryl groupsoptionally comprising at least one hetero atom selected from halogens,N, O, and S. Examples of moieties comprising at least one bond linkagethat comprises at least one hetero atom of high electronegativity or Sinclude, but are not limited to moieties comprising at least one amidebond, urethane bond, urea bond, thiourea bond, ether bond, or thioetherbond.

A¹, A², A³, A⁴, A⁵, A⁶, A⁷, A⁸, A⁹, and A¹⁰, which may be the same ordifferent, may, independently, be NABEGs selected from moietiescomprising at least one bond linkage that comprises at least one heteroatom of high electronegativity or S; and aryl groups optionallycomprising at least one hetero atom selected from halogens, N, O, and S.Examples of moieties comprising at least one bond linkage that comprisesat least one hetero atom of high electronegativity or S include, but arenot limited to moieties comprising at least one amide bond, urethanebond, urea bond, thiourea bond, ether bond, or thioether bond. A¹, A²,A³, A⁴, A⁵, A⁶, A⁷, A⁸, A⁹, and A¹⁰ may be such that BRIDGE comprises atmost one positive charge, or is substantially neutral, and in the lattercase, each of these constituents may itself be substantially neutral,which includes actual neutrality.

BRIDGE may comprise any suitable number of non-hydrogen atoms, aspreviously described, such as from about 10 to about 100 non-hydrogenatoms, inclusive, merely by way of example.

Merely by way of example, BRIDGE may have the formula (Formula 2) setforth directly below.—(CH₂)_(x)—C(═O)NH—(CH₂)_(α)—[O—(CH₂)_(β)]_(b)—[O—(CH₂)_(γ)]_(c)—NH(O═C)—(CH₂)_(x)—  Formula2In one such case, for example, each L of BRIDGE is —(CH₂)_(x)—, whereeach x, independently, is an integer selected from 1 to 11, inclusive;A¹ of BRIDGE is —C(═O)NH—; a of BRIDGE is 1; A² of BRIDGE is —O—; A³ ofBRIDGE is —O—; α may be an integer selected from 2 to about 20,inclusive; each of β and γ, independently, may be 2 or 3; b may be zeroor an integer selected from 2 to about 20; and c may be zero or 1; eachof d, e, f, g, h and i of BRIDGE is 0; and A¹⁰ of BRIDGE is —NH(O═C)C—.Merely by way of example, BRIDGE may be as just described, wherein cis 1. Further, merely by way of example, BRIDGE may be as justdescribed, wherein c is 1, and further, wherein x may be 5; α and γ maybe the same and may be 2 or 3; β may be 2; and b may be 0, 1, 2 or 3.

Each of the constituent monomeric dyes, Q1 and Q2, of the dimeric dye isa fluorescent nucleic acid dye. In general, Q1 and Q2 are selected andcovalently linked via BRIDGE in a manner to encourage or to ensureintramolecular dimer formation in the absence of DNA and formation ofhighly fluorescent DNA-dye complexes upon DNA binding. A dimeric dye mayhave a tendency to form an intramolecular dimer as may be associatedwith the formation of a useful hairpin-like structure, as previouslydescribed. Such a dimeric dye may possess desirable properties, such aslow background fluorescence and low toxicity, for example.

Intramolecular dimer formation may be confirmed by comparing absorptionspectra of a dimeric dye in an aqueous solution and absorption spectraof the related monomeric dye or dyes also in an aqueous solution. Anyintramolecular dimer formation should cause the spectra of the componentmonomeric dyes in the dimeric dye to be shifted significantly relativeto the spectra of the related monomeric dye(s). In this regard, asignificant shift may be about 10 nm or more, by way of example. Forexample, in FIG. 2, the spectra associated with AOAO-7 are shiftedsignificantly relative to the spectra of DMAO.

When the intramolecular dimer formation is an H-dimer formation, thespectra will usually undergo a significant blue shift. In this regard, asignificant shift may be about 10 nm or more, by way of example. Othertypes of intramolecular dimer formation are also possible and may resultin spectral shift in another direction, in insignificant spectral shift,or the like. In this regard, an insignificant shift may be about 5 nm orless, by way of example. Additional analytical methods for detectingintramolecular dimer formation may include nuclear magnetic resonance(NMR), infrared spectroscopy (IR), or the like. Any intramolecular dyeaggregation that results in a hairpin structure is generally desirable.

Various combinations of Q₁ and Q₂ may be useful or desirable. By way ofexample, examples of dimeric dyes and associated intermediates arelisted below in Table 1.

TABLE 1 Prepared Fluorescent Nucleic Acid Dyes SPACER LENGTH No. NameStructure MWt. (ATOMS)  1 DMAO

478.41 N/A  2 TMAO

620.35 N/A  3 AO-3N

705.16 N/A  4 AO-2N

493.43 N/A  5 PMAO

691.47 N/A  6 AOAO-1

926.76 10  7 AOAO-2

1124.03 21  8 AOAO-3

1038.88 16  9 AOAO-2Q

1252.71 11 10 AOAO-4

1041.95 14 11 AOAO-5

896.73 8 12 AOAO-6

1010.92 16 13 AOAO-7

1080.96 19 14 TOTO-3

1088.94 16 15 AOAO-8

1064.92 16* 16 AOAO-9

1229 25 17 AOAO-10

1313.24 31 18 AOAO-11

1123 22 19 AOAO-12

1082.94 19 20 AOAO-13

1215.14 27 21 AOAO-14

1621.61 53 22 AOAO-12R

1132.95 19 23 AOTO-3

1094.99 16 24 TOTO-12

1146.23 20 25 TO(3)TO(3)-12

1245.34 20 26 TO(3)TO(3)-2

1302.44 22 27 AORO-7

1320.25 21 28 RORO-12

1550.51 22 29 TOTO-13

1248 27 30 STST-27

1116 27 31 STST-19

1000.8 19 32 AOAO-47

1547.6 47 33 AOAO-67

1864 67 34 AOAO-113

2541 113 35 ET-27

1239 27 36 STST-21N

1041 21

While many of the structures shown in Table 1 show one or more iodideanion(s), any other appropriate anion(s), such as those describedherein, such as chloride anion(s), merely by way of example, may be usedin place of the iodide anions shown.

A dimeric dye may comprise a fluorescent nucleic acid dye Q₁ and afluorescent nucleic acid dye Q₂, wherein Q₁ and Q₂ may be the same, ordifferent. A dimeric dye may comprise a pair of identical fluorescentmonomeric nucleic acid dyes. When Q₁ and Q₂ are the same, the resultingdye is a homodimer, such as any of Dye Nos. 6-22, 24-26, and 28-36 ofTable 1, merely by way of example. When Q₁ and Q₂ are differentfluorescent nucleic acid dyes that have similar absorption and emissionspectra, the resulting dimer is a heterodimer, such as that of Dye No.23 of Table 1, merely by way of example. Such a heterodimer isfunctionally similar to a homodimer. In either of the foregoing cases,both Q₁ and Q₂ are reporter dyes, such that upon DNA binding, they bothcontribute to the detected fluorescent signal. When Q₁ and Q₂ aredifferent fluorescent nucleic acid dyes that have substantiallydifferent absorption and emission spectra, the resulting dimer is aheterodimer. In this latter case, only one of the two dyes, Q₁ and Q₂,is selected as a reporter dye.

Fluorescent nucleic acid dyes and examples thereof are now described.Examples of a monomeric fluorescent nucleic acid dye suitable forconstructing dimeric dyes include, but are not limited to, an acridinedye, an asymmetric cyanine-based nucleic acid stain, a phenanthridiniumdye, a symmetric cyanine nucleic stain, a pyronin dye, a styryl dye, aderivative of DAPI, and a derivative of a Hoechst dye. DAPI and Hoechstdyes generally cannot be directly attached to BRIDGE because they do notpossess a reactive group for bond formation. In this context, aderivative refers to a base dye, such as DAPI or a Hoechst dye, that ismodified sufficiently for bond formation, such as by addition of areactive group, by way of example.

The monomeric fluorescent nucleic acid dye may be an acridine dye havingthe general structure (Structure 2) set forth directly below.

Acridine orange (AO) is an acridine dye that stains dsDNA with greenfluorescence and stains RNA with red fluorescence. Traganos, et al., J.Histochem. Cytochem. 25(1), 46 (1977). Unlike some other acridine dyes,AO has a high extinction coefficient (>50,000) and a long absorptionwavelength (λ_(abs)=500 nm (DNA bound)). However, the affinity of AO fornucleic acid is very low and the dye has significant intrinsicfluorescence in the absence of nucleic acids. In this regard, the levelof intrinsic fluorescence may be significant in that it precludes thedye from being used in detecting nucleic acid at a low level, such as inthe low nanogram/mL range, for example, or in detecting nucleic acid ingels without a destaining step, for example. Consequently, AO itself isof little utility for DNA or RNA quantification.

An acridine dye may comprise any of a variety of substituents at variouspositions on the ring structure. The nature of a substituent and itssubstitution position may strongly affect the spectral properties of thedye produced. In general, electron-donating substituents at the 3- and6-positions and an electron-withdrawing substituent at the 9-positiontypically red-shift the absorption and emission spectra of the dye.Examples of a typical electron-donating group include, but are notlimited to, an amino group, a hydroxyl group, an alkoxy group, and analkylmercapto group. Examples of a typical electron-withdrawing groupinclude, but are not limited to, a cyano group, a perfluoroalkyl group,a carboxamido group, a sulfonamide group, a nitro group, and a halogengroup. Any additional ring structure fused with the core structure willalso increase the wavelengths of the dye produced.

Various portions of Structure 2 are now described. In Structure 2, as invarious other monomeric dye structures provided or described herein, asymbol of “R” followed by a subscript, such as R₁, merely by way ofexample, may indicate a substituent of the structure that is not part ofBRIDGE, or may represent where BRIDGE attaches to the structure, inwhich case, it is not a substituent of the structure. Each R₁,independently, may be H; an alkyl or alkenyl having 1 carbon to 6carbons, inclusive; a halogen; —OR₄; —SR₅; —NR₆R₇; —CN; —NH(C═O)R₈;—NHS(═O)₂R₉; or —C(═O)NHR₁₀; and any adjacent pair of R₁s optionallyform a 5- or 6-membered saturated or unsaturated ring, which furtheroptionally comprises at least one hetero atom selected from N, O and S.One of the R₁s may represent where BRIDGE attaches to the structure, inwhich case, that R₁ is merely representative and not actually asubstituent of the monomeric dye. In any case where R₁ involves at leastone of R₄, R₅, R₆, R₇, R₈, R₉, and R₁₀, any applicable one of same isindependently H or an alkyl having 1 carbon to 6 carbons, inclusive, andfor any applicable pair of adjacent R₆ and R₇, independently, R₆ and R₇may in combination form a 5- or 6-membered saturated or unsaturatedring, which optionally comprises at least one hetero atom selected fromN and O.

Typically, R₂ is H; an alkyl or alkenyl having 1 carbon to 6 carbons,inclusive; an aryl optionally comprising at least one hetero atomselected from halogens, N, O and S; a halogen; —OR₁₁; —SR₁₂; —NHR₁₃;—CN; or —C(═O)NHR₁₄; or represents where BRIDGE attaches to thestructure. In any case where R₂ involves at least one of R₁₁, R₁₂, R₁₃and R₁₄, any applicable one of same is independently H or alkyl having 1carbon to 6 carbons, inclusive.

Typically, R₃ is H; or an alkyl having 1 carbon to 6 carbons, inclusive;or represents where BRIDGE attaches to the structure.

Ψ is an anion, such as an anion that balances positive charge(s)associated with the dye, for example. Ψ may be biologically compatible.Examples of a suitable anion include, but are not limited to, a halide,a sulfate, a phosphate, a perchlorate, a tetrafluoroborate, and ahexafluorophosphate. Merely by way of example, the anion may be chlorideor iodide.

Only one of R₁, R₂ and R₃ must represent where BRIDGE attaches to thestructure. Merely by way of example, one of R₂ and R₃ may representwhere BRIDGE attaches to the structure. As described herein, BRIDGE maybe covalently linked to a monomeric acridine dye, such as any such dyedescribed herein, and to another suitable monomeric dye, to form adimeric dye.

The monomeric acridine dye may have the structure (Structure 3) setforth directly below.

In Structure 3, generally, each R₁, independently, is H, or a C1-C2,inclusive, alkyl; one of R₂ and R₃ represents where BRIDGE attaches tothe structure; when R₂ represents where BRIDGE attaches to thestructure, R₃ is H or —CH₃; when R₃ represents where BRIDGE attaches tothe structure, R₂ is selected from H, —CH₃, —NH₂, —NHCH₃, —CN, and—C(═O)NH₂; each of R₆ and R₇, independently, is H, or a C1-C2,inclusive, alkyl; and Ψ is an anion, as previously described. Merely byway of example, for each pair of adjacent R₆ or R₇ and R₁,independently, R₆ or R₇ and R₁ may in combination form a 5- or6-membered, saturated or unsaturated ring. Further, merely by way ofexample, two monomeric acridine dye molecules of Structure 3 incombination with BRIDGE of Formula 2 may form a dimeric dye.

In one example, the monomeric acridine dye, as represented by Structure3, may be such that each R₁ is H; R₂ is H; R₃ represents where BRIDGEattaches to the structure; each R₆ is —CH₃; each R₇ is —CH₃; and Ψ is ananion, as previously described.

A dimeric dye, such as a dimeric acridine dye, for example, may beuseful for detecting nucleic acids immobilized relative to a matrix,such as a solid matrix, a semi-solid matrix, or a gel matrix, forexample, or a surface, such as a solid surface, a membrane surface, aglass surface, a plastic surface, or a polysilicon surface, for example.A dimeric acridine dye may be useful for nucleic acid gel staining, suchas pre-cast nucleic acid gel staining or post-nucleic acid gel staining.In such an application, there is no need for a de-staining step. Ingeneral, nucleic acid gel staining using a dimeric acridine dye isassociated with relatively high sensitivity and relatively lowbackground fluorescence. In this regard, sensitivity generally refers toan ability to detect a low level of nucleic acids, as shown by thenumber and brightness of bands appearing on the right-side lanes of thegel, such as the gel of Photograph C of FIG. 8, for example, and anability to detect short nucleic acid fragments, as shown by the numberand relative brightness of the bands appearing on the lower portion ofthe gel of Photograph C of FIG. 8, for example; and low backgroundfluorescence generally refers to an ability to detect nucleic acidpresence without having to destain the gel.

In an example (Example 50), each of three separate dyes, SYBR Safe, SYBRGreen I, and Dye No. 20 of Table 1, a dimeric acridine dye, was preparedand used in post-DNA gel staining. Three separate photographs associatedwith the use of the three separate dyes, respectively, are shown in FIG.8. These photographs demonstrate that Dye No. 20 is substantially moresensitive than SYBR Safe and is about as sensitive as SYBR Green I.However, SYBR Green I is known to be hydrolytically unstable in aqueoussolution, and particularly, in buffers having a pH of 7 or more. Karsaiet al., Biotechniques 32(4), 790 (2002). Dye No. 20 is relatively stablein water or in buffers that are commonly used in connection gelelectrophoresis, at room temperature for about 3 months or more.

The monomeric fluorescent nucleic acid dye may be an asymmetric cyaninedye having the general structure (Structure 4) set forth directly below.

The general structure (Structure 4, above) of an asymmetric cyanine dyemay be divided into three parts: 1) a heterocyclic ring that is asubstituted benzazolium ring; 2) a methane or polymethine bridge; and 3)a heterocyclic ring that is a substituted pyridinium or quinoliniumring. The dotted line in the structure represents the atoms necessary toform one or more fused aromatic ring(s), optionally incorporating one ormore nitrogen(s), which may or may not be quaternized. When the dottedline represents a 6-membered ring comprising one or more nitrogenatom(s), the resulting fused ring is called an aza-benzole ring.

In Structure 4, in general, each of R₁ and R₁′ on the benzazolium ring,independently, is H; alkyl or alkenyl having 1 carbon to 6 carbons,inclusive; a halogen; —OR₉, —SR₁₀; —NR₁₁R₁₂; —CN; —NH(C═O)R₁₃;—NHS(═O)₂R₁₄; or —C(═O)NHR₁₅. Merely by way of example, one of R₁ andR₁′ may be a substituent that is meta to X or to the benzazole nitrogen,wherein the substituent confers at least one desirable property asfurther described below. In any case where R₁ or R₁′ involves at leastone of R₉, R₁₀, R₁₁, R₁₂, R₁₃, R₁₄ and R₁₅, any applicable one of same,independently, is H; or alkyl having 1 carbon to 12 carbons, inclusive,optionally incorporating 1 to 2 nitrogen(s), inclusive; or an aryl; andany applicable R₁₁ and R₁₂ may in combination form a 5- or 6-memberedsaturated or unsaturated ring, which optionally comprises at least onehetero atom selected from N and O.

As mentioned above, one of R₁ and R₁′ of Structure 4 may be asubstituent that confers at least one desirable property to the dye. Onesuch desirable property is DNA minor groove-binding. A minorgroove-binding molecule typically has a structure with a crescent shapethat fits into the minor groove of a double-stranded DNA. Examples of aDNA minor groove-binding dye molecule or non-dye molecule, which mayinclude a natural molecule, include, but are not limited to, DAPI, aHoechst dye, distamycin A, netropsin, and any of numerous syntheticminor groove-binders based on polyamides of N-methylpyrrole andN-methylimidazole. Catalog of Biotium, Inc. (Hayward, Calif. (CA)),2005-2006; Boger, et al., Acc. Chem. Res. 37, 61 (2004); and Dervan, P.B., Bioorg. & Med. Chem. 9, 2215 (2001). The crescent shape of a minorgroove-binder is typically created by meta-substitution of a 5- or6-membered ring with a minor groove-binder substituent, which includes,but is not limited to, a substituted or an unsubstitutedbenzoxazol-2-yl, a substituted or an unsubstituted benzimidazol-2-yl, asubstituted or an unsubstituted benzothiazol-2-yl, a substituted or anunsubstituted imidazol-2-yl, a substituted or an unsubstitutedoxazol-2-yl, a substituted or an unsubstituted thiazol-2-yl, asubstituted or an unsubstituted N-methylpyrrolyl-2-aminocarbonyl, asubstituted or an unsubstituted N-methylpyrrolyl-3-carboxamido, asubstituted or an unsubstituted 1-methylimidazol-2-carboxamido, asubstituted or an unsubstituted 1-methylimidazol-4-aminocarbonyl, asubstituted or an unsubstituted phenyl, a substituted or anunsubstituted pyridyl, a substituted or an unsubstituted pyrazinyl, anda substituted or an unsubstituted triazinyl. A DNA dye may bemeta-substituted by a minor groove-binder substituent as described inU.S. Patent Application Publication No. 2004/0132046.

One of R₁ and R₁′ may represent where BRIDGE attaches to the structure.

X is selected from O and S. In general, a dye wherein X is S has longerabsorption and emission wavelengths than a similar dye wherein X is O.

R₂ may be methyl or ethyl, or may represent wherein BRIDGE attaches tothe structure. Merely by way of example, R₂ may be methyl or ethyl.

The subscript n represents a number of double bond units in any methinebridge and is selected from 0, 1, and 2. Typically, a dye with a longermethine bridge will have longer wavelengths than a dye with a shortermethine bridge. Merely by way of example, n may be 0 or 1.

Substitutents R₃, R₄, and R₅ are independently H or —CH₃. Optionally,any adjacent pair of these substitutents may form a 5- or 6-memberedring. Merely by way of example, R₃, R₄, and R₅ may be H.

Independently, each of substituents R₆, R₈, and R_(9′) may be H; analkyl or alkenyl having 1 carbon to 10 carbons, inclusive, optionallycomprising at least one hetero atom selected from N, O, and S; ahalogen; —OR₁₆; —SR₁₆; —NR₁₆R₁₇; or a substituted or unsubstituted aryl,optionally comprising 1 to 3 hetero atom(s), inclusive, selected fromhalogens, N, O, and S. R₈ and R_(8′) may in combination form a fusedaromatic ring, which may be further substituted 1 to 4 time(s),inclusive, independently, by C1-C2, inclusive, alkyl, C1-C2, inclusive,alkoxy, C1-C2, inclusive, alkylmercapto, or a halogen. In any case inwhich any of R₆, R₈, and R_(8′) involve at least one of R₁₆ and R₁₇, anyapplicable one of same, independently, is H; or alkyl having 1 carbon to12 carbons, inclusive, optionally incorporating 1 to 2 nitrogen(s),inclusive; or an aryl; and any applicable R₁₆ and R₁₇ may in combinationform a 5- or 6-membered saturated or unsaturated ring, which optionallycomprises at least one hetero atom selected from N and O.

R₆ may represent where BRIDGE attaches to the structure.

R₇ is selected from H; an alkyl or alkenyl having 1 carbon to 10carbons, inclusive, optionally comprising an aryl and at least onehetero atom selected from N, O, and S; or a substituted or unsubstitutedaryl optionally comprising 1 to 3 hetero atom(s), inclusive, selectedfrom halogens, N, O, and S; or may represent where BRIDGE attaches tothe structure.

Ψ is an anion, as previously described herein.

Only one of R₁, R₁′, R₆, R₇ and R₉ must represent where BRIDGE attachesto the structure. As described herein, BRIDGE may be covalently linkedto a monomeric asymmetric cyanine dye, such as any such dye describedherein, and to another suitable monomeric dye, to form a dimeric dye.

An asymmetric cyanine dye may have the structure (Structure 5) set forthdirectly below, wherein each of R₁ ¹, R₆, R₇, R₈ and R_(8′) is aspreviously described in connection with Structure 4.

By way of example, the asymmetric cyanine dye, as represented byStructure 5, may be such that R₁′ is H; alkyl or alkenyl having 1 carbonto 6 carbons, inclusive; a halogen; —OR₉; —SR₁₀; —NR₁₁R₁₂; —CN;—NHS(═O)₂R₁₄; —C(═O)NHR₁₅; or a substituent associated with minor groovebinding; or represents where BRIDGE attaches to the structure. Further,when R₁′ comprises at least one of R₉, R₁₀, R₁₁, R₁₂, R₁₃, R₁₄ and R₁₅,any said one of R₉, R₁₀, R₁₁, R₁₂, R₁₃, R₁₄ and R₁₅, independently, is Hor alkyl having 1 carbon to 12 carbons, inclusive, optionallyincorporating 1 to 2 nitrogen(s), inclusive, or an aryl; and when R₁′comprises R₁₁ and R₁₂, R₁₁ and R₁₂ may in combination form a 5- or6-membered, saturated or unsaturated ring, which optionally comprises atleast one hetero atom selected from N and O. X may be selected from Oand S and n may be selected from 0, 1, and 2. R₆ may be H; alkyl oralkenyl having 1 carbon to 10 carbons, inclusive, optionally comprisingat least one hetero atom selected from N, O, and S; a halogen; —OR₁₆;—SR₁₆; —NR₁₆R₁₇; or a substituted or an unsubstituted aryl, optionallycomprising 1 to 3 hetero atom(s), inclusive, selected from halogens, N,O, and S; or may represent where BRIDGE attaches to the structure. R₇may be H; alkyl or alkenyl having 1 carbon to 10 carbons, inclusive,optionally comprising an aryl and at least one hetero atom selected fromN, O, and S; or a substituted or an unsubstituted aryl optionallycomprising 1 to 3 hetero atom(s), inclusive, selected from halogens, N,O, and S; or may represent where BRIDGE attaches to the structure. R₈may be H; alkyl or alkenyl having 1 carbon to 10 carbons, inclusive,optionally comprising at least one hetero atom selected from N, O, andS; a halogen; —OR₁₆; —SR₁₆; —NR₁₆R₁₇; or a substituted or anunsubstituted aryl, optionally comprising 1 to 3 hetero atom(s),inclusive, selected from halogens, N, O, and S; or may represent whereBRIDGE attaches to the structure. R₈′ may be H; alkyl or alkenyl having1 carbon to 10 carbons, inclusive, optionally comprising at least onehetero atom selected from N, O, and S; a halogen; —OR₁₆; —SR₁₆;—NR₁₆R₁₇; or a substituted or an unsubstituted aryl, optionallycomprising 1 to 3 hetero atom(s), inclusive, selected from halogens, N,O, and S. R₈ and R₈′ may in combination form a fused aromatic ring,which may be further substituted 1 to 4 time(s), inclusive,independently, by C1-C2, inclusive, alkyl, C1-C2, inclusive, alkoxy,C1-C2, inclusive, alkylmercapto, or a halogen. For any R₆, R₈, or R₈′that comprises at least one of R₁₆ and R₁₇, any said one of R₁₆ and R₁₇thereof, independently, may be H; alkyl having 1 carbon to 12 carbons,inclusive, optionally incorporating 1 to 2 nitrogen(s) or an aryl. Forany R₆, R₈, and R₈′ that comprises R₁₆ and R₁₇, R₁₆ and R₁₇ thereof mayin combination form a 5- or 6-membered saturated or unsaturated ring,which optionally comprises at least one hetero atom selected from N andO. Only one of R₁′, R₆, R₇ and R₈ represents where BRIDGE attaches tothe structure. Ψis an anion, as previously described.

An asymmetric cyanine dye may have the structure (Structure 6) set forthdirectly below, wherein R₇ represents where BRIDGE attaches to thestructure and is Ψ an anion, as previously described.

Merely by way of example, two monomeric asymmetric cyanine dye moleculesof Structure 6 in combination with BRIDGE of Formula 2 may form adimeric dye.

A dimeric asymmetric cyanine dye may be useful for detecting nucleicacids immobilized relative to a matrix or a surface, as previouslydescribed in connection with another dye, or the like, and for nucleicacid gel staining, such as pre-cast nucleic acid gel staining or postnucleic acid gel staining. In general, nucleic acid gel staining using adimeric cyanine dye is associated with high sensitivity and lowbackground fluorescence. In such an application, there is no need for ade-staining step. In an example (Example 50) described herein, each ofthree separate dyes, SYBR Safe, SYBR Green I, and Dye No. 29 of Table 1,a dimeric asymmetric cyanine dye, was prepared and used in post-DNA gelstaining. Three separate photographs associated with the use of thethree separate dyes, respectively, are shown in FIG. 9. Thesephotographs demonstrate that Dye No. 29 is substantially more sensitivethan SYBR Safe and is as sensitive as SYBR Green I. In this regard,sensitivity generally refers an ability to detect low levels of nucleicacids and/or short nucleic acid fragments, as previously describedherein. Unlike SYBR Green 1, Dye No. 20 of Table 1 is relatively stableis in buffers that are commonly used in connection gel electrophoresis,such as TBE, at room temperature for about 3 months or more.

A dimeric asymmetric cyanine dye comprising monomeric asymmetric cyaninedyes of Structure 6 may be excited by UV light or by visible light, suchas the blue light equipped in a Dark Reader transilluminator from ClareChemical Research (Dolores, Colo.) and a 488 nm argon laser equipped insome of the commercial laser-based gel scanners, for example. In anexample (Example 50) described herein, Dye No. 29 of Table 1 wasprepared and used in post-DNA gel staining. A photograph of associatedwith the use of Dye No. 29 is shown in FIG. 11. Relatively new gelreaders, such as the Dark Reader transilluminator, which use a visiblelight source, have been developed as a safer alternative to traditionalUV light-based transilluminators. These alternative gel readers mayemploy a blue light with a peak centered around 470 nm. There are alsogel readers that use light from 488 nm argon lasers. A gel stain mustabsorb light sufficiently within a wavelength range of 460-510 nm,inclusive, in order to be read using the various visible light-based gelreaders. As such, a gel stained with EB cannot be read using a visiblelight-based gel reader because the absorption peak of EB is not withinthe appropriate wavelength range. Dye No. 29 of Table 1 has a verystrong and broad absorption or excitation peak centered around 500 nm,as demonstrated in relation to Example 51 and graphically shown in FIG.10, such that a gel stained with this dye can be read using a visiblelight-based gel reader, as demonstrated in relation to Example 50 andshown in FIG. 11.

The monomeric fluorescent nucleic acid dye may be a phenanthridiniumderivative, having the general structure (Structure 7) set forthdirectly below.

In general, R₁ may represent where BRIDGE attaches to the structure,although it will be understood that many variations of Structure 7 aboveare possible and contemplated herein, via a variety of techniques, suchas synthesis techniques that may provide for the attachment of BRIDGE tothe structure elsewhere or that may modify the structure to provide adye with any of various desirable wavelengths. Ψ is an anion, aspreviously described.

Merely by way of example, two monomeric phenanthridinium dye moleculesof Structure 7 in combination with BRIDGE of Formula 2 may form adimeric dye.

A dimeric phenanthridinium dye may be useful for detecting nucleic acidsimmobilized relative to a matrix or a surface, as previously describedin relation to another dye, or the like, and for nucleic acid gelstaining, such as pre-cast nucleic acid gel staining or post-nucleicacid gel staining. In general, nucleic acid gel staining using a dimericphenanthridinium dye is associated with high sensitivity and lowbackground fluorescence. In such an application, there is no need for ade-staining step. In an example (Example 49) described herein, each oftwo separate dyes, EB and Dye No. 35 of Table 1 (ET-27), a dimericphenanthridinium dye, was prepared and used in pre-cast DNA gelstaining. Photographs associated with the use of the two dyes,respectively, are shown in FIG. 12.

A dimeric phenanthridinium dye may be relatively stable, evenexceptionally so, relative to an SYBR dye, such as those mentionedherein, which may be relatively unstable. In an example (Example 52)described herein, each of two separate dyes, SYBR Gold and Dye No. 35 ofTable 1 (ET-27), a dimeric phenanthridinium dye, was prepared andmonitored in terms of optical density. A graphical representation ofnormalized absorbance versus time for each of the solutions is shown inFIG. 13. As shown, the absorbance associated with Dye No. 35 wasrelatively constant over the 24-hour period, while the absorbance ofSYBR Gold decreased by nearly 50% over the same period. Dye No. 35 isgenerally stable in an electrophoresis buffer, such as TBE buffer, atroom temperature for at least about 6 months. Further, this dye in TBEbuffer may be heated in a microwave oven for at least about 10 minuteswithout decomposition.

In a gel electrophoresis application, an agarose gel may be prepared byheating (via microwave, for example) a suspension of agarose powder inan electrophoresis buffer, such as TBE buffer, thereby producing a hotagarose solution, pouring the solution onto a slab, and cooling thesolution, thereby producing a useful gel. In a pre-cast gel stainingapplication, an EB dye may be added directly to the agarose powdersuspension prior to the heating of the suspension, as EB is generallysufficiently stable, both hydrolytically and thermally. This isgenerally not the case with a SYBR Green dye, as such a dye is generallyof limited hydrolytic and thermal stability. For example, in a pre-castgel staining application, SYBR Green I dye is generally combined with anagarose solution after the initial agarose suspension has been heatedand the resulting agarose solution has been cooled as much as possible.Combining the SYBR Green I dye with the agarose solution in this mannermay be a delicate task, as if the temperature of the agarose solution istoo high, the dye my decompose, and if the temperature of the agarosesolution is too low, the agarose solution may gel up such that the dyeand the solution are inadequately combined or mixed. Further by way ofexample, in a pre-cast gel staining application, a pre-cast gel preparedwith SYBR Green I is generally used within 24 hours, as it may loseutility thereafter. As SYBR Gold is even less stable than SYBR Green, itgenerally cannot be used to make a precast gel. Dye No. 35 of Table 1 isof sufficient hydrolytic and thermal stability to be used in thepreparation of a pre-cast gel, such as a pre-cast gel that may be storedfor at least about 3 months without loss of performance, for example.This dye may offer sufficient to exceptional stability, as well as lowor minimal background fluorescence relative to EB and low or minimaltoxicity relative to EB, as further described below.

Dye No. 35 of Table 1 is relatively low in toxicity and mutagenicity. Amutagenicity comparison was made between Dye No. 35 and EB using aChromoPlate test kit from EBPI (Brampton, Ontario, Canada). The test kitis based on the Ames Test, a bacterial reverse mutation test. The testemploys a mutant strain, or several strains, of Salmonella typhimuriumbacteria, carrying mutation(s) in the operon coding for histidinebiosynthesis. When the bacteria are exposed to mutagenic agents undercertain conditions, reverse mutation from amino acid (histidine)auxotrophy to prototrophy occurs. In an example (Example 53) describedherein, Dye No. 35 and EB were assayed using three dose levels (0.25nmole, 2.5 nmoles and 25 nmoles), respectively, for each dye, and in theabsence and presence of S9 extract for each dose level and each dye,respectively. S9 extract is a rat liver extract that comprises variousmetabolic enzymes. The tests associated with the presence of S9 extractwere undertaken to give information on the potential genotoxicity of themetabolized dyes. Bacterium strain TA98, a frame shift indicator, wasused for the tests (according to a protocol provided by the supplier)since EB is a known frame shift mutagen as shown by Ames Test using thesame bacterial strain. Each single test was carried out using 36 wellsand the number of positive wells out of each 36 wells was taken as anindicator of the relative mutagenicity levels of the dye under the testconditions. As a negative control, bacteria were also incubated in theabsence of a dye. The results are shown in Table 2 below.

TABLE 2 Mutagenicity Data Without S9 extract Positive wells Sample Exp 1Exp 2 Control 3 0 EB (0.25 nmole) 0 0 EB (2.5 nmoles) 0 3 EB (25 nmoles)0 3 Dye No. 35 (0.25 nmole) 2 0 Dye No. 35 (2.5 nmoles) 1 3 Dye No. 35(25 nmoles) 1 3 With S9 extract Positive Sample wells Control 4 EB (0.25nmole) 22 EB (2.5 nmoles) 17 EB (25 nmoles) 16 Dye No. 35 (0.25 nmole) 0Dye No. 35 (2.5 nmoles) 9 Dye No. 35 (25 nmoles) 0

As shown in Table 2, relative to the negative control in which no dyewas present, both Dye No. 35 and EB showed very weak mutagenicity in theabsence of S9 extract. As also shown, EB showed significantly greatermutagenicity than Dye No. 35 in the presence of S9 extract. It isbelieved that the relatively low mutagenicity of Dye No. 35 may beattributable to a combination of factors, such as the high molecularweight of the dye, where the high molecular weight makes it difficultfor the dye to enter cells, and the H-dimer formation property of thedye, where the H-dimer formation lowers the effective concentration ofthe dye.

The monomeric fluorescent nucleic acid dye may be a xanthene derivative,having the general structure (Structure 8) set forth directly below.

Certain cationically charged xanthene dyes are known to bind to nucleicacids. For example, pyronin Y, in which R₁, R₂, and R₂′ are methyl andR₃, R₃′, R₄, R₄′, and A are H, is a known fluorescent DNA binding dyethat has been used for DNA gel staining. Adkin et al., Anal. Biochem.240(1), 17 (1996). A dye having the general skeleton shown in Structure8 above is expected to have similar nucleic acid staining properties andto provide other fluorescent colors. For example, pyronin Y has anabsorption maximum at 548 nm and an emission maximum at 565 nm,providing a red fluorescent color.

Merely by way of example, in general, each of R₁, R₂, R₁′, and R₂′,independently, may be H, or C1-C6, inclusive, alkyl, optionallyincorporating 1 to 2 hetero atom(s) selected from N and O. Furthermerely by way of example, independently, at least one of the pair R₁ andR₂ and the pair R₁′ and R₂′ may in combination form a 5- or 6-memberedring, optionally comprising one hetero atom selected from N and O. R₁and R₁′ may be the same and R₂ and R₂′ may be the same.

One of R₁, R₂, R₁′, and R₂′ may represent where BRIDGE attaches to thestructure. It may be that one of R₁, R₂, R₁′, R₂′ and A may representwhere BRIDGE attaches to the structure.

Merely by way of example, R₃, R₃′, R₄, and R₄′, independently, may be Hor C1-C3, inclusive, alkyl. R₃, R₃′, R₄, and R₄′ may be the same.Independently, at least one of the pair R₃ and R₁, the pair R₂ and R₄,the pair R₃′ and R₁′, and the pair R₄ and R₂′ may in combination form a5- or 6-membered ring, which may be saturated or unsaturated,substituted or unsubstituted.

A is a C1-C3, inclusive, alkyl, or represents where BRIDGE attaches tothe structure.

Ψ is an anion, as previously described.

Two monomeric xanthene dye molecules of Structure 8 in combination withBRIDGE of Formula 2 may form a dimeric dye.

Other monomeric fluorescent nuclei acid stains, such as DAPI, DIPI, aHoechst dye, LDS 751, hydroxystilbamidine, a styryl dye, a merocyaninedye, a cyanine dye, or FluoroGold, merely by way of example, may besuitable for use or may be derivatized to be suitable for use asdescribed herein. Haugland, R.P., Handbook of Fluorescent Probes andResearch Products, 9^(th) edition. It will be understood that a largenumber of other monomeric nucleic acid dyes may be suitable for use ormay be derivatized to be suitable for use as described herein. The dyesmay either be directly conjugated to BRIDGE or be derivatized so thatthey can be conjugated to BRIDGE using synthesis knowledge.

Dimeric nucleic acid dyes may be useful for detecting nucleic acidsimmobilized relative to a matrix or a surface, as previously describedherein in connection with various dyes, or as nucleic acid gel stains.

There are generally two methods for staining nucleic acids in gels usinga fluorescent nucleic acid dye. The first method is post-gel staining,wherein a nucleic acid sample is separated by gel electrophoresis, thegel comprising the separated nucleic acids is bathed in a solutioncomprising the dye, the gel may be destained, if desirable or necessaryto remove background fluorescence, and the resulting gel is viewedand/or documented using a transilluminator and/or a photographingdevice. The second method is pre-cast gel staining, wherein a gel ispremixed or pre-embedded with the dye, the nucleic acid sample isseparated by electrophoresis using the pre-cast gel, and the stained gelis viewed and/or documented using a transilluminator and/or aphotographing device. In general, a dimeric nucleic acid dye can be usedfor post-gel staining, pre-cast gel staining, or variations thereof. Assuch a dye is generally associated with low background fluorescence,destaining is usually not required.

Post-nucleic acid gel staining may be carried out using a dimericnucleic acid dye. Generally, post-nucleic acid gel staining may comprisepreparation of a gel, electrophoretic separation of a nucleic acidsample, preparation of a dye solution, staining of the gel comprisingseparated nucleic acid molecules, and/or visualization of the stainedgel, as now generally described.

The gel may be prepared by a known method or any appropriate method. Thegel may be an agarose gel, a polyacrylamide gel, or the like. Ingeneral, the density of a gel, which is determined by the amount ofagarose or acylamide monomer per volume unit, affects DNA migration, andthus separation of DNA bands. By way of example, for suitable or optimalDNA band resolution, a gel with a relatively lower percentage ofagarose, such as 0.3-0.7% (weight/volume), may be used for relativelylonger DNA samples, such as DNA of 5-60 kb. Further by way of example, agel with a relatively higher percentage of agarose, such as 1.5-3%(weight/volume), may be used for relatively shorter DNA fragments, suchas DNA of 0.1 to 1 kb. In general, an agarose gel comprising about 1% ofagarose may be used for separating DNA of relatively common lengths orsizes, such as from about 0.5 to about 6 kb.

A nucleic acid sample may be prepared, loaded onto a gel, and thenseparated by electrophoresis. The process may involve the preparation ofa dye stock solution. A dye stock solution may be prepared by dissolvinga solid dimeric dye in an aqueous solvent, such as water or a buffer ora water-miscible organic solvent, such as DMSO or DMF. In general, theconcentration of the stock solution is about 100-fold to about10,000-fold greater relative to the concentration of a working dyesolution. The working dye solution may be prepared by diluting the dyestock solution to an effective dye concentration with an aqueous solventselected from water; a solution of at least one salt that comprises ananion that is associated with a strong acid and a cation that isassociated with a strong base, such as a salt previously describedherein, for example; and a buffer selected from phosphate bufferedsaline (PBS), tris acetate (TAE), and tris borate (TBE); and anycombination thereof. Merely by way of example, the dye stock solutionmay be so diluted in an aqueous solution comprising a salt thatcomprises an anion that is associated with a strong acid and a cationthat is associated with a strong base. Examples of such a salt include,but are not limited to, sodium chloride, sodium bromide, sodium sulfate,potassium chloride, potassium bromide, potassium sulfate, magnesiumchloride, and tetramethylammonium chloride. In such a case, the saltconcentration may be from about 5 mM to about 0.5 M, inclusive, about0.05 M to about 0.2 M, inclusive, or about 0.1 M. In general, aneffective working concentration of the dye is from about 0.1 μM to about100 μM, inclusive, or from about 0.5 μM to about 10 μM, inclusive.

The gel and the working dye solution may be placed in contact in anappropriate manner, such as by immersing the gel in the working dyesolution for a sufficient amount of time, such as from about 10 to about90 minutes, inclusive, or about 30 minutes. Once the gel is stained bythe working dye solution, the stained gel may be illuminated with alight of suitable wavelength to generate a fluorescence signal.Equipment useful for illuminating a stained gel includes, but is notlimited to, a UV transilluminator, a laser scanner, a Dark Reader fromClare Chemical Research, Inc. (Dolores, Colo.), or the like, merely byway of example. The fluorescence signal may be detected in anyappropriate manner, such as via visual inspection, a camera, aphotographic film, or the like, merely by way of example.

Pre-cast nucleic acid gel staining may be carried out using a dimericnucleic acid dye. Generally, this pre-cast nucleic acid gel staining maycomprise preparation and casting of a stained gel, electrophoreticseparation of a nucleic acid sample, and/or visualization of the stainedgel, as now generally described.

A gel solution comprising a dimeric dye may be prepared in various ways.A dye stock solution may be prepared as generally described above inrelation to post-gel staining. In one example, a gel solution comprisinga dimeric dye may be prepared by combining or mixing an aliquot of thedye stock solution with a suitable amount of agarose powder in anelectrophoresis buffer, such as TBE buffer, and heating the resultingsolution under conditions suitable for producing an approximatelyhomogeneous gel solution, such as via a microwave oven, for example, ata suitable setting or temperature, for example, and for a sufficientamount of time, such as a few minutes, for example. In another example,a gel solution comprising a dimeric dye may be prepared by preparing anagarose gel solution as just described, but without the dye, andcombining or mixing the resulting solution with an aliquot of the dyestock solution sufficient to provide an effective working concentrationof the dye. In either example or any suitable preparation, the effectiveworking concentration of the dye in the gel solution may generally befrom about 0.1 μM to about 100 μM, inclusive, or from about 0.5 μM toabout 10 μM, inclusive. Further, in either example or any suitablepreparation, the amount of agarose in the gel solution may generally befrom about 0.5% to about 8%, inclusive. Other gel solutions, such as gelsolutions comprising a suitable component other than agarose, may besimilarly prepared.

A gel may be cast using the heated gel solution. A nucleic acid samplemay be prepared, loaded onto the gel, and then electrophoreticallyseparated in a known manner or any appropriate manner. The gelcomprising the separated nucleic acid sample may be illuminated in anyappropriate manner to generate a fluorescence signal, which may then bedetected or visualized in any appropriate manner, such as that generallydescribed above in connection with post-gel staining.

Any suitable variation of the above-described nucleic acid detectionschemes may be employed and is contemplated herein. Merely by way ofexample, a nucleic acid sample to be detected may be present and/orimmobilized in a sieving matrix, in a sedimentation or buoyant densitygradient, on an inert matrix such as a blot, on a testing strip, on anyother solid or semi-solid support, or the like.

A post-gel staining solution may comprise an aqueous solution thatcomprises a nucleic acid stain and at least one salt that comprises ananion that is associated with a strong acid and a cation that isassociated with a strong base. The concentration of the stain relativeto the solution may be from about 0.1 μM to about 100 μM, inclusive, orfrom about 0.5 μM to about 10 μM, inclusive, for example. The stain ordye component may be any useful staining dye, such as a monomeric ordimeric nucleic acid dye, merely by way of example. The salt may have aconcentration of from about 5 mM to about 0.5 M, inclusive, about 0.05 Mto about 0.2 M, inclusive, or about 0.1 M, for example. The saltcomponent may enhance, perhaps significantly so, the staining quality ofa post-nucleic acid staining solution. The salt may be selected from thesodium chloride, sodium bromide, sodium sulfate, potassium chloride,potassium bromide, potassium sulfate, tetramethylammonium chloride,and/or magnesium chloride for example. The aqueous solution may furthercomprise, optionally, a buffer.

A post-gel staining solution comprising a salt that comprises an anionthat is associated with a strong acid and a cation that is associatedwith a strong base provides better results in a nucleic acidgel-staining application than does a post-gel staining solution thatcomprises a buffer, but no such salt. This holds true when the post-gelstaining solution comprises a dimeric nucleic acid dye, such as thosedescribed herein, or a monomeric nucleic acid dye, such as an asymmetriccyanine nucleic acid dye, such as SYBR Green I, SYBR Safe, SYBR Gold andGelStar, merely by way of example.

In an example (Example 49) described herein, each of two separate dyes,SYBR Green I and Dye No. 29 of Table 1, a dimeric asymmetric cyaninedye, was prepared and used in post-DNA gel staining. Three separatephotographs (A, B and C) associated with the use of three separate anddifferent preparations of the SYBR Green I dye, and three separatephotographs (D, E and F) associated with the use of three separate anddifferent preparations of Dye No. 29, respectively, as described in thebrief description of FIG. 14 herein, are shown in FIG. 14. Photographs Aand D are associated with dye preparations comprising a buffer, but noseparate salt, and photographs B, C, E and F are associated with dyepreparations comprising a salt, NaCl. These photographs demonstrate thatgel staining solutions prepared with a salt comprising an anion that isassociated with a strong acid and a cation that is associated with astrong base, such as NaCl, may provide superior nucleic acid stainingresults relative to gel staining solutions prepared with a buffer andwithout such a salt. For example, as shown in photographs B, C, E and Fof FIG. 14, gels stained with a staining solution comprising NaCl wereassociated with relatively brighter DNA bands overall and with DNA bandsof decreasing brightness from the first lane on the left (greater DNAloading) to the 4th lane on the right (lesser DNA loading), as expected.As shown in photograph A of FIG. 14, the gel stained with SYBR Green Istaining solution that lacked NaCl was associated with relatively weakeror less bright DNA bands overall and with DNA bands that were relativelyunreflective of the relative amount of DNA loaded in each lane, as shownby the more or less evenly weak signals in the left three lanes.

A dimeric nucleic acid dye may be included in a kit. A kit may comprisethe dye, information or a protocol regarding use of the dye or the kit,and/or other useful or necessary materials or reagents, such as anymaterials or reagents suitable for the detection of nucleic acids, forexample, such as a buffer, a DNA or RNA ladder, and/or agarose, forexample. A kit may comprise the dye impregnated into paper, such aspaper provided by Edvotek (Bethesda, Md.) or disclosed in EuropeanPatent Office Publication No. EP 1 057 001 A2 or World IntellectualProperty Organization International Publication No. WO 99/42620.

A dimeric nucleic acid dye may be synthesized via synthesis of monomericdye constituents, synthesis of BRIDGE, and conjugation of the monomericdye constituents to BRIDGE. Syntheses of monomeric dyes and monomericdyes comprising a functional group or a reactive group are nowdescribed.

Suitable monomeric dyes and monomeric dyes comprising a functional groupor a reactive group may be prepared from scratch by a known procedure orany suitable procedure, or by modifying commercially available materialthat already has a suitable or desirable core structure. Many monomericacridine dyes may be prepared from commercially available acridine dyes.A few examples of a commercially available acridine dye that may serveas suitable starting material for synthesis are set forth directlybelow.

Other acridine core structures may be prepared according to knownprocedures or any suitable procedures. Albert, A., The acridines: theirpreparation, physical, chemical, and biological properties and uses,Edward Arnold Ltd., London; Eldho, et al., Synth. Commun. 29, 4007(1999); and Joseph, et al., Bioconjugate Chem. 15, 1230 (2004). Anacridine core structure may be formed by condensing a suitablediphenylamine with a suitable carboxylic acid or a carboxylic acidequivalent in the presence of a Lewis acid, as schematically illustratedin Reaction 1 directly below.

In Reaction 1, R′, R″ and R′″ are suitable substituents, as furtherdescribed below. The diphenylamine starting material is eithercommercially available or may be synthesized from a suitable arylhalideand a suitable arylamine using a known method or any suitable method.Yang, et al., J. Organomet. Chem. 576, 125 (1999); Hartwig, et al., J.Org. Chem. 64, 5575 (1999); and Wolfe, et al., J. Org. Chem. 65, 1158(2000).

The nature of the substituents and the position where the substituentsare attached may have a profound effect on the spectral property of thedye. In general, electron-donating groups at the 2-, 3-, 6- and 7-positions will increase the absorption and emission wavelengths of thedye. A typical electron-donating group may be an amino group, analkylamino group, a dialkylamino group, a hydroxyl group, an alkoxygroup, a thiol group, or an alkylthio group, by way of example. A moretypical electron-donating group may be an amino group, an alkylaminogroup, a dialkylamino group, or an alkoxy group, by way of example. Ingeneral, an electron-withdrawing group at the 9-position will increasethe absorption and emission wavelengths of the dye. A typicalelectron-withdrawing group may be a cyano group, a perfluoroalkyl group,an aminocarbonyl group, an alkylaminocarbonyl group, an alkylcarbonylgroup, an aldehyde group, an alkoxycarbonyl group, an aminosulfonatogroup, an alkylaminosulfonato group, or a halide group, by way ofexample. A more typical electron-withdrawing group may be a cyano group,a perfluoroalkyl group, or a halide group.

In general, once the acridine core structure is built, the 10-nitrogenis alkylated with a haloalkyl group, which typically comprises anadditional reactive group or a functional group that can be converted toa reactive group. The additional reactive group serves to conjugate theacridine dye to BRIDGE. Several ways of making monomeric acridine orangedyes with a suitable reactive group are schematically illustrated inScheme 1 directly below.

The 9-position of 10-alkylated acridine may be substituted with a cyanogroup, which may be further hydrolyzed to a carboxamide group, asschematically illustrated in Scheme 2 directly below.

Methods of preparing reactive monomeric asymmetric cyanine dyes havebeen described. Carreon, et al., Org. Lett. 6(4), 517 (2004). Such a dyemay have the structure (Structure 9) set forth directly below.

U.S. Pat. No. 5,863,753 discloses the preparation of a series ofreactive asymmetric cyanine dyes, including ones that have a substituentortho to the quinolinium or pyridinium nitrogen. Such a substituent,especially a cyclic substituent, ortho to the quinolinium or pyridiniumnitrogen, is said to confer desired properties to the asymmetric cyaninedyes, according to U.S. Pat. No. 5,436,134. These cyclically substitutedasymmetric cyanine dyes are commonly referred to as SYBR dyes. Zipper,et al., Nucleic Acids Res. 32(12), e103 (2004). Some of the reactiveSYBR dyes are commercially available from Molecular Probes, Inc.(Eugene, Oreg.), although the exact structures of these dyes are notknown. Haugland, R.P., Handbook of Fluorescent Probes and ResearchChemicals, 9^(th) edition.

U.S. Patent Application Publication No. 2004/0132046 discloses methodsfor preparing monomeric asymmetric cyanine dyes with minorgroove-binding capability. In general, these dyes possess acrescent-shaped structure by virtue of having an additional benzazolylsubstitutent on the benzazolyl ring of the dyes. Similar monomeric dyeshaving a suitable reactive group may be prepared using similar methods,for example, as schematically illustrated in Scheme 3 directly below.

Reactive phenanthridinium dyes may be prepared from the commerciallyavailable 3,8-diamino-6-phenylphenanthridine, as schematicallyillustrated in Scheme 4 directly below.

Preparations of pyronin derivatives with a reactive group at the9-position may be carried out by condensing two equivalents ofm-aminophenol derivative with one equivalent of dicarboxylic anhydride,as schematically illustrated in Scheme 5 directly below.

Many monomeric non-fluorescent nucleic acid-binding dyes are knownpigments used in textile and ink industries and are commerciallyavailable. References for preparations of these dyes can be found in theliterature. Many suitable reactive monomeric fluorescent non-nucleicacid dyes and non-fluorescent non-nucleic acid dyes are commerciallyavailable or may be prepared using known methods.

BRIDGE is usually formed when the monomeric dyes are coupled to abi-functional group, which is often commercially available. In general,the terminal portions of BRIDGE are from the monomeric dyes themselves,while the middle portion of BRIDGE is from a bi-functional moleculeavailable from a commercial source. In some cases, a portion or asignificant portion of BRIDGE, such as up to about 90%, for example, maybe pre-attached to the monomeric dyes prior to the final assembly of thedimeric dye. In some other cases, most of BRIDGE may be preparedseparately before the monomeric dyes are attached. In the case ofheterodimer synthesis, a mono-protected bi-functional linker group isusually first attached to one monomeric dye, followed by de-protectionand coupling to the second monomeric dye.

In general, dimeric may be assembled by conjugating monomeric dyeshaving a suitable reactive group with a bi-functional linker in aone-step coupling reaction for some of the homodimers, or in multi-stepreactions for heterodimers or some of the homodimers comprising multiplebridge element A. Examples of synthetic routes to selected homodimer andheterodimers are schematically illustrated in Scheme 6 directly below.

EXAMPLES Example 1 Preparation of 10-(3-Iodopropyl)acridine Orange,Iodide

One equivalent of 1,3-diiodopropane was added to a suspension of 5 g ofacridine orange (Aldrich) in 10 mL of chlorobenzene. The resultingmixture was stirred at 90-100° C. overnight. The hot reaction mixturewas poured into ˜200 mL of EtOAc. The orange precipitate was collectedby filtration and dried under vacuum, yielding ˜8 g.

Example 2 Preparation of 10-(5-Carboxypentyl)acridine Orange, ChlorideSalt

10-(5-Ethoxycarbonylpentyl)acridine bromide was prepared using theprocedure of Example 1, with the exception that 1,3-diiodopropane wasreplaced with ethyl 6-bromohexanoic acid. The crude product (5 g) wassuspended in ˜100 mL methanol and 3 equivalents of NaOH dissolved in 30mL H₂O. The suspension was stirred at room temperature for 24 h.Methanol was removed by evaporation, and the remaining aqueous solutionwas acidified with concentrated HCl. About 50 mL saturated NaCl wasadded to precipitate the product. The product was collected byfiltration and then dried under vacuum at 45° C. for 24 hours.

Example 3 Preparation of DMAO (Dye No. 1 of Table 1)

10-(3-Iodopropyl)acridine orange, iodide (100 mg) was suspended in 20 mL2M dimethylamine in methanol in a sealed tube and then stirred at 60° C.overnight. The mixture was cooled to room temperature and then pouredinto 50 mL EtOAc. The precipitate was collected by centrifugation andthen dried under vacuum at 40° C. for 24 hours.

Example 4 Preparation of TMAO (Dye No. 2 of Table 1)

A mixture of DMAO (Dye No. 1 of Table 1) (11 mg, 0.023 mmol) and CH₃I(0.5 mL) in CH₃OH (2 mL) was refluxed gently for 4 days. The orangeproduct (10 mg) was collected by suction filtration.

Example 5 Preparation of PMAO (Dye No. 5 of Table 1)

A mixture of 10-(3-iodopropyl)acridine orange iodide salt (100 mg, 0.18mmol) and N,N,N′N′-tetramethyl-1,3-propanediamine (0.3 mL, 1.8 mmol) inCH₃OH (10 mL) was refluxed overnight. After cooling down to roomtemperature, the precipitate was collected by suction filtration. Theprecipitate was resuspended in CH₃OH (5 mL) and refluxed overnight andcollected by suction filtration. It was dried to a constant weight invacuo to give a dark red solid (14 mg).

Example 6 Preparation of AOAO-2Q (Dye No. 9 of Table 1)

A mixture of 10-(3-iodopropyl)acridine orange iodide salt (81 mg, 0.15mmol) and PMAO (100 mg, 0.15 mmol) in DMF (1.5 mL) was heated at 130° C.for 7 hours. After cooling down to room temperature, CH₃OH (15 mL) wasadded and the suspension was heated to reflux for 1 hour. Suctionfiltration gave the product as dark red solid (83.1 mg).

Example 7 Preparation of AOAO-2 (Dye No. 7 of Table 1)

Et₃N (0.15 mL, 1.05 mmol) and TSTU (320 mg, 1.05 mmol) were added to asuspension of 10-(5-carboxypentyl)acridine orange chloride salt (438 mg,1.03 mmol) in DMF (5 mL) at room temperature. The mixture was stirred atroom temperature for 15 minutes, followed by the addition of Et₃N (0.1mL) and 3,3′-diamino-N-methyldipropylamine (50 mg, 0.344 mmol). Afterthe mixture was stirred at room temperature overnight, EtOAc (20 mL) wasadded to precipitate the product. The crude product was re-dissolved inDMF and precipitated out again with EtOAc. The solid (250 mg) wasseparated by centrifugation.

Example 8 Preparation of AOAO-3 (Dye No. 8 of Table 1)

The dye (393 mg) was prepared by using the procedure to synthesizeAOAO-2 from 10-(5-carboxypentyl)acridine orange (432 mg, 1.03 mmol) andethylenediamine (25 mg, 0.42 mmol).

Example 9 Preparation of 10-(8-Bromooctyl)acridine Orange Bromide

A mixture of acridine orange (2 g, 7.53 mmol) and 1,8-dibromoactane (12mL, 67.8 mmol) in chlorobenzene (15 mL) was heated at 110° C. overnight.EtOAc (50 mL) was added and the suspension was refluxed for 1 hour.After cooling down to room temperature, suction filtration gave theproduct as orange solid (3.56 g).

Example 10 Preparation of AOAO-5 (Dye No. 11 of Table 1)

A mixture of 10-(8-bromoactyl)acridine orange bromide (0.5 g, 0.94 mmol)and acridine orange (0.3 g, 11.2 mmol) in DMF (5 mL) was heated at 130°C. overnight. EtOAc was added to precipitate the product. Repeatprecipitate from DMF and EtOAc gave the product as dark red solid (214mg).

Example 11 Preparation of AOAO-7 (Dye No. 13 of Table 1)

The dye (30 mg) was synthesized by using the procedure to make AOAO-2from 10-(5-carboxypentyl)acridine orange chloride salt (121 mg, 0.29mmol) and 1,5-diamino-pentane dihydrochloride (20 mg, 0.12 mmol).

Example 12 Preparation of AOAO-8 (Dye No. 15 of Table 1)

The dye (182 mg) was synthesized by using the procedure to make AOAO-2from 1045-carboxypentyl)acridine orange chloride salt (241 mg, 0.58mmol) and piperazine (20 mg, 0.23 mmol).

Example 13 Preparation of AOAO-11 (Dye No. 18 of Table 1)

The dye (112 mg) was synthesized by using the procedure to make AOAO-2from 10-(5-carboxypentyl)acridine orange chloride salt (180 mg, 0.43mmol) and 1,8-diamino-octane (25 mg, 0.17 mmol).

Example 14 Preparation of AOAO-12 (Dye No. 19 of Table 1)

The dye (76 mg) was synthesized by using the procedure to make AOAO-2from 1045-carboxypentyl)acridine orange chloride salt (147 mg, 0.35mmol) and 2,2′ oxybis(ethyl-amine) dihydrochloride (25 mg, 0.14 mmol).

Example 15 Preparation of AOAO-13 (Dye No. 20 of Table 1)

The dye (64 mg) was synthesized by using the procedure to make AOAO-2from 10-(5-carboxypentyl)acridine orange chloride salt (96 mg, 0.23mmol) and 4,7,10-trioxa-1,13-tridecanediamine (20 mg, 0.09 mmol).

Example 16 Preparation of1,3-Di-((2-(N-t-Boc-amino)ethyl)aminocarbonyl)benzene (Dye No. 101,Shown Directly Below)

Et₃N (0.4 mL, 2.71 mmol) and TSTU (820 mg, 2.71 mmol) were added to asolution of isophthalic acid (220 mg, 1.32 mmol) in DMF (2 mL) at roomtemperature. The mixture was stirred at room temperature for 30 minutes.Addition of Et₃N (1 mL) and mono-tBoc-ethylenediamine (460 mg, 2.86mmol) followed. The mixture was stirred at room temperature overnightand then partitioned between 1N HCl (100 mL) and EtOAc (50 mL). Theaqueous layer was extracted with EtOAc (50 mL) and the combined organiclayers were washed with 1N HCl (2×50 mL), H₂O (50 mL), and saturatedNaCl (50 mL), and dried with anhydrous Na₂SO₄. The crude product waspurified by column chromatography using EtOAc:hexanes (9:1) as eluent togive the colorless solid product (356 mg).

Example 17 Preparation of 1,3-Di-((2-aminoethyl)aminocarbonyl)benzene,Trifluoro-acetic Acid Salt (Dye No. 102, Shown Directly Below)

1,3-di-((2-(N-t-Boc-amino)ethyl)aminocarbonyl)benzene (356 mg, 0.79mmol) was added to TFA (5 mL) at 0° C. The mixture was stirred at 0° C.for 1 hour and the solution was concentrated to dryness in vacuo. Thecolorless residue was dissolved in CH₃OH (2 mL) and added dropwise toEt₂O (30 mL). The precipitate was collected by centrifugation and driedto a constant weight in vacuo to give the solid product (425 mg).

Example 18 Preparation of AOAO-9 (Dye No. 16 of Table 1)

The dye (55 mg) was prepared by using the procedure to make AOAO-2 from10-(5-carboxypentyl)acridine orange chloride salt (109 mg, 0.26 mmol)and 1,3-Di((2-aminoethyl)aminocarbonyl)benzene, trifluoroacetic acidsalt (50 mg, 0.1 mmol).

Example 19 Preparation of1,3-Di((5-(N-t-Boc-amino)pentyl)aminocarbonyl)benzene (Dye No. 103,Shown Directly Below)

The dye (555 mg) was prepared according to the procedure to make1,3-di-((2-(N-t-Boc-amino)ethyl)aminocarbonyl)benzene from isophthalicacid (254 mg, 1.53 mmol) and mono-tBoc cadaverine (640 mg, 3.15 mmol).

Example 20 Preparation of 1,3-Di-((5-aminopentyl)aminocarbonyl)benzene,trifluoro-acetic Acid Salt (Dye No. 104, Shown Directly Below)

The dye (560 mg) was prepared according to the procedure for Dye No. 102(555 mg, 1.04 mmol).

Example 21 Preparation of AOAO-10 (Dye No. 17 of Table 1)

The dye (22 mg) was prepared by using the procedure to make AOAO-2 from10-(5-carboxypentyl)acridine orange chloride (95 mg, 0.23 mmol) and DyeNo. 104 (50 mg, 0.09 mmol).

Example 22 Preparation of AOAO-14 (Dye No. 21 of Table 1)

The dye (150 mg) was prepared by using the procedure to make AOAO-2 from10-(5-carboxypentyl)acridine orange chloride (166 mg, 0.40 mmol) anddiamido-dPEG-diamine (Quanta Biodesign of Powell, Ohio) (100 mg, 0.15mmol).

Example 23 Preparation of10-(((3-(N-Boc-amino)propyl)-N,N-dimethyl)ammonium)propyl) Acridine,Diiodide (Dye No. 105, Shown Directly Below)

A mixture of 10-(3-iodopropyl)acridine orange iodide (500 mg, 0.89 mmol)and 3-(N-t-Boc-amino)propyl-N,N-dimethylamine (1.8 g, 8.9 mmol) in CH₃OH(50 mL) was refluxed overnight. After cooling down to room temperature,the precipitate was collected by suction filtration and dried to aconstant weight to give Dye No. 105 as an orange solid (292 mg).

Example 24 Preparation of10-((((3-ammonium)propyl)-N,N-dimethyl)ammonium)propyl Acridine,Trifluoroacetate Salt (Dye No. 106, Shown Directly Below)

Dye No. 105 (50 mg, 0.06 mmol) was added to TFA (2 mL) at 0° C. Themixture was stirred at 0° C. for 30 minutes. The solution wasconcentrated to dryness in vacuo and the residue was dissolved in CH₃OH(3 mL). The solution was added dropwise to Et₂O (30 mL) and theprecipitate was collected by centrifugation and dried to a constantweight in vacuo to give Dye No. 106 as an orange solid (28 mg).

Example 25 Preparation of AOAO-4 (Dye No. 10 of Table 1)

The dye (23 mg) was prepared by using the procedure to make AOAO-2 from10-(5-carboxypentyl)acridine orange chloride salt (31 mg, 0.075 mmol)and Dye No. 106 (28 mg, 0.036 mmol).

Example 26 Preparation of 10-(6-(N-Phthalimido)hexyl)acridine OrangeBromide Salt (Dye No. 107, Shown Directly Below)

A mixture of acridine orange (2 g, 7.54 mmol) andN-(6-bromohexyl)phthalimide (4.7 g, 15.1 mmol) in chlorobenzene (20 mL)was heated at 110° C. for 2 days. EtOAc (50 mL) was added and thesuspension was heated to reflux for 1 hour. After cooling down to roomtemperature, the product Dye No. 107 was collected by suction filtrationas an orange solid (3.76 g).

Example 27 Preparation of10-(5-((5-Carboxypentyl)aminocarbonyl)pentyl)acridine Orange, Iodide(Dye No. 108, Shown Directly Below)

Et₃N (40 μL, 0.28 mmol) and TSTU (81 mg, 0.27 mmol) were added to asuspension of 10-(5-carboxypentyl)acridine orange chloride (107 mg,0.258 mmol) in DMF (3 mL). The mixture was stirred at room temperaturefor 15 minutes. Addition of Et₃N (0.2 mL) and a solution of6-aminohexanoic acid (67 mg, 0.51 mmol) in H₂O (1 mL) followed. Themixture was stirred at room temperature for 1 hour and concentrated todryness in vacuo. The residue was triturated with H₂O to give Dye No.108 as an orange solid (125 mg).

Example 28 Preparation of 9-Cyano-10-(5-carboxypentyl)acridine Orange,Chloride (Dye No. 109, Shown Directly Below)

A mixture of 10-(5-carboxypentyl)acridine orange (0.15 g, 0.361 mmol)and sodium cyanide (35 mg, 0.722 mmol) in DMF (3 mL) was stirred at roomtemperature in open air for 2 days. CH₃CN (10 mL) was added and theresulting suspension was stirred at room temperature for 1 hour. Thedark blue precipitate was collected by centrifugation and dried to aconstant weight in vacuo to give Dye No. 109 (130 mg).

Example 29 Preparation of AOAO-12R (Dye No. 22 of Table 1)

Et₃N (32 μL, 0.23 mmol) and TSTU (68 mg, 0.227 mmol) were added to asolution of Dye No. 109 (98.3 mg, 0.223 mmol) in DMF (2 mL) at roomtemperature. The mixture was stirred at room temperature for 15 minutes.Addition of Et₃N (100 μL) and 2,2′-oxybis-(ethylamine) dihydrochloride(16 mg, 0.09 mmol) followed. The mixture was stirred at room temperaturefor 2 days. The solution was concentrated to about 1 mL and EtOAc (2 mL)was added. The precipitate was collected by centrifugation. The productwas re-dissolved in DMF and precipitated again with EtOAc to give DyeNo. 22 as a dark blue solid (54.4 mg).

Example 30 Preparation of 9-Aminocarbonyl-10-(5-carboxypentyl)acridine(Dye No. 110, Shown Directly Below)

A solution of Dye No. 109 (30 mg, 0.068 mmol) in 75% H₂SO₄ (1 mL) washeated at 60° C. for 2 days. After cooling down to room temperature, themixture was added to Et₂O (10 mL). The precipitate was collected bycentrifugation and re-dissolved in CH₃OH (1.5 mL). EtOAc (10 mL) wasadded and the solid precipitate was collected by centrifugation anddried to a constant weight in vacuo to give Dye No. 110 as a dark pinksolid (20.4 mg).

Example 31 Preparation of N-Carboxypentyl Thiazole Orange (ShownDirectly Below)

The dye was prepared using published procedure (Carreon, et al., Org.Let. 6(4), 517 (2004)).

Example 32 Preparation of TOTO-3 (Dye No. 14 of Table 1)

The dye (354 mg) was prepared using the procedure to synthesize AOAO-2from N-Carboxypentyl thiazole orange (460 mg, 1.04 mmol) and ethylenediamine (25 mg, 0.42 mmol).

Example 33 Preparation of10-(542-(N-t-Boc-amino)ethyl)aminocarbonyl)pentyl)acridine OrangeChloride Salt (Dye No. 111, Shown Directly Below)

Et₃N (106 μL, 0.76 mmol) and TSTU (230 mg, 0.76 mmol) were added to asuspension of 10-(5-carboxypentyl)acridine orange chloride (302 mg, 0.73mmol) in DMF (3 mL). The mixture was stirred at room temperature for 15minutes. The addition of Et₃N (350 μL) and mono t-BOC-ethylenediamine(150 mg, 0.92 mmol) followed. The mixture was stirred at roomtemperature for 1 hour and then concentrated to dryness in vacuo. Theresidue was dissolved in CH₃CN (2 mL) and precipitated by the additionof EtOAc (20 mL). The precipitate was collected by centrifugation anddried to a constant weight to give Dye No. 111 as orange solid (365 mg).

Example 34 Preparation of10-(5-((2-Ammoniumethyl)aminocarbonyl)pentyl)acridine Orange,Trifluoroacetate (Dye No. 112, Shown Directly Below)

Dye No. 111 (347 mg, 622 mmol) was added in one portion totrifluoroacetic acid (3 mL) at 5° C. The mixture was stirred at 5° C.for 1 hour and concentrated to dryness in vacuo. The residue wasdissolved in CH₃OH (3 mL) and added dropwise to Et₂O (50 mL). Theprecipitate was collected by centrifugation to give Dye No. 112 asorange solid (297 mg).

Example 35 Preparation of AOTO-3 (Dye No. 23 of Table 1)

Et₃N (20 μL, 0.142 mmol) and TSTU (42.2 mg, 0.142 mmol) were added to asolution of N-carboxypentylthiazole orange (62 mg, 0.142 mmol) in DMF (1mL). The mixture was stirred at room temperature for 15 minutes.Addition of Et₃N (70 μL) and Dye No. 112 (50 mg, 0.095 mmol) followed.The mixture was stirred at room temperature for 2 hours and thenconcentrated to dryness in vacuo. The residue was re-dissolved in DMF (1mL) and EtOAc (2 mL) was added. The precipitate was collected bycentrifugation. Repeated precipitation from DMF and EtOAc gave theproduct as orange red solid (50.4 mg)

Example 36 Preparation of TOTO-12 (Dye No. 24 of Table 1)

The dye (19.4 mg) was prepared by using the procedure to synthesizeAOAO-2 from N-carboxypentylthiazole orange (94.5 mg, 0.2145 mmol) and2,2′ oxybis(ethylamine) dihydrochloride (15 mg, 0.085 mmol).

Example 37 Preparation of TO(3)TO(3)-12 (Dye No. 25 of Table 1)

The dye (32.6 mg) was prepared by using the procedure to synthesizeAOAO-2 from N-carboxypentyl thazole blue (Carreon, et al., Org. Let.6(4), 517 (2004); and Benson, et al., Nucleic Acid Res. 21(24), 5727(1993)) (99 mg, 0.212 mmol) and 2,2′ oxybis(ethylamine) dihydrochloride(15 mg, 0.085 mmol).

Example 38 Preparation of TO(3)TO(3)-2 (Dye No. 26 of Table 1)

The dye (28.4 mg) was prepared by using the procedure to synthesizeAOAO-2 from N-carboxypentyl thiazole blue (76 mg, 0.173 mmol) and3,3′-diamino-N-methyldi-propylamine (10 mg, 0.069 mmol).

Example 39 Preparation of10-(5-((5-(N-t-Boc-amino)pentyl)aminocarbonyl)pentyl)acridine Orange,Chloride (Dye No. 113, Shown Directly Below)

The dye (280 mg) was prepared by using the procedure to synthesize DyeNo. 111 from 10-(5-carboxypentyl)acridine orange chloride (200 mg, 0.483mmol) and mono t-BOC-cadaverine (130 mg, 0.628 mmol).

Example 40 Preparation of10-(5((5-ammoniumpentyl)aminocarbonyl)pentyl)acridine Orange,Trifluoroacetate (Dye No. 114, Shown Directly Below)

Dye No. 114 (234 mg) was prepared by using the procedure to synthesizeDye No. 112 from Dye No. 113 (280 mg, 0.467 mmol).

Example 41 Preparation of AORO-7 (Dye No. 27 of Table 1)

The compound (29 mg) was prepared by using the procedure to synthesizeAOTO-3 from compound No. 114 (35 mg, 0.06 μmol) and the rosamine dye(Biotium, Inc. (Hayward, Calif.)) (40 mg, 0.063 mmol).

Example 42 Preparation of TOTO-13 (Dye No. 29 of Table 1)

The compound (102 mg) was prepared by using the procedure to synthesizeAOAO-2 (Dye No. 7 of Table 1) from N-carboxypentyl thiazole orange (102mg, 0.23 mmole) (Example 31) and 4,7,10-trioxa-1,13-tridecanediamine (23mg, 0.1 mole).

Example 43 Preparation ofN-(5-carboxypentyl)-4-(4-(dimethylamino)styryl)pyridinium Bromide

A mixture of 4-N,N-dimethylaminobenzaldehyde (3 g, 20 mmoles),N-(5-carboxypentyl)picolinium bromide (5.6 g, 20 mmoles) and piperidine(2 mL) in ethanol (100 mL) was heated at 60° C. overnight. The mixturewas evaporated to dryness in vacuo. The residue was redissolved inmethanol and then precipitated with ether to give the title product (6.7g).

Example 44 Preparation of STST-19 (Dye No. 31 of Table 1)

The dye (85 mg) was prepared by using the procedure to make AOAO-2(Example 7) fromN-(5-carboxypentyl)-4-(4-(dimethylamino)styryl)pyridinium bromide(Example 43) (200 mg, 0.5 mmole) and 2,2′-oxybis(ethylamine)dihydrochloride (36 mg, 0.2 mmoles).

Example 45 Preparation of STST-27 (Dye No. 30 of Table 1)

The dye (81.8 mg) was prepared by using the procedure to make AOAO-2(Example 7) fromN-(5-carboxypentyl)-4-(4-(dimethylamino)styryl)pyridinium bromide(Example 43) (200 mg, 0.5 mmole) and 4,7,10-trioxa-1,13-tridecanediamine(44 mg, 0.2 mmoles).

Example 46 Absorbance and Fluorescence of DMAO and AOAO-7

The absorbance spectra, as shown in FIG. 2 and FIG. 3, and fluorescenceemission spectra, as shown in FIG. 4, of DMAO and AOAO-7, were measuredseparately without DNA presence, or with DNA presence (2 mg/ml of salmonsperm DNA), in PBS buffer. All dye concentrations were adjusted toprovide an optical density of 0.05 at 495 nm. The spectra werenormalized to 1 in the absorbance plot. Relative to DMAO, AOAO-7exhibits a new shorter wavelength peak at 471 nm in absorbance,indicating aggregation of the two acridine monomers within AOAO-7. Uponbinding to DNA, absorbances of AOAO-7 and DMAO showed 5 nm- and 10nm-red shifts, respectively, relative to free dyes. The fluorescence offree AOAO-7 is about 5 times lower than that of DMAO. The fluorescenceper acridine monomer of AOAO-7 is close to that of DMAO, indicating thattwo monomers of AOAO-7 no longer quenched each other when bound to DNAand the linker between the two did not exhibit negative effect on thequantum yield.

Example 47 Absorbance Spectra of TOTO-1 and TOTO-3

In a similar manner to that described in connection with Example 46, theabsorbance spectra of TOTO-1 and TOTO-3 were measured without DNApresence, as shown in FIG. 5, or in the presence of 2 mg/ml of salmonsperm DNA, as shown in FIG. 6, in PBS buffer. As shown in FIG. 5, thespectra of the free dyes indicate that TOTO-3, which has BRIDGE that isneutral or substantially devoid of positive charges, forms anintramolecular H-dimer, or a hairpin structure, while TOTO-1, which hasmultiple positive charges; has less spectral shift. As shown in FIG. 6,the absorption spectra of both TOTO-1 and TOTO-3 in the presence of DNAshift to about the same position, indicating that the hairpin structureof TOTO-3 dimer opens up upon binding to DNA, and that both TOTO-1 andTOTO-3 form similar types of DNA-dye complexes.

Example 48 Fluorescence of AOAO-12 in response to different amount ofDNA

The fluorescence of 0.1 μM AOAO-12 in 200 mL of PBS in the presence of0, 0.1, 0.2, 0.4, 0.6, 0.8, 1.0, 2.0, 4.0, 6.0, 8.0 and 10 μg/ml finalconcentrations of salmon sperm DNA or a mixture of single-stranded 20meroligonucleotide were measured on a microtiter plate reader (SpectraMaxof Molecular Devices Corporation (Sunnyvale, Calif.)). The fluorescencewas plotted against DNA concentration, as shown in FIG. 7. It can beseen that fluorescence linearly responded to DNA up to 2.0 μg/ml(inset). At higher concentrations of DNA, the response becamenon-linear. AOAO-12 fluoresces more intensely when bound to doublestranded DNA than when bound to single stranded DNA.

Example 49 Pre-Cast DNA Gel Staining

An agarose gel solution (1% agarose) was prepared following a standardprotocol (J. Roskams et al., Lab Ref A Handbook of Recipes, Reagents,and Other Reference Tools for Use at the Bench, Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y., 2002). A stock solution of aDye No. 35 (ET-27) of Table 1 in DMF at 12 mM concentration wasprepared. An aliquot of the stock solution of dye was added to the gelsolution, while it was hot, resulting in an effective workingconcentration of the dye of about 1.2 μM. The resulting solution wasthoroughly mixed by swirling. The resulting gel solution was poured ontoa gel slab to cast the gel. Serial two-fold dilutions of 1 kb Plus DNALatter from Invitrogen Co. (Carlsbad, Calif.) were made and theresulting DNA samples were loaded onto the gel in four lanes from leftto right with a loading of 200 ng, 100 ng, 50 ng, and 25 ng per lane,respectively. The DNA samples were electrophoretically separated in1×TBE buffer using a standard protocol. The resulting gel was thenviewed using a UV transilluminator with 300 nm excitation. The sameprocedure was followed using a stock solution of EB at about 0.5 μg/mL(or 1.3 μM) in DMF in place of the stock solution of Dye No. 35 of Table1.

Photographs of the illuminated gels were taken with an EB filter andPolaroid 667 black-and-white film, as shown in FIG. 12. The resultsdemonstrate that relative to EB, Dye No. 35 is more sensitive withrespect to shorter DNA fragments, as shown by the number and thebrightness of the bands appearing in the lower portion of the left-mostphotograph versus those appearing in the lower portion of the right-mostphotograph, and more sensitive with respect to low level DNA, as shownby the number and the brightness of the bands appearing in theright-side lanes of the left-most photograph versus those appearing inthe right-side lanes of the right-most photograph. It should be notedthat other dyes described herein may be prepared and employed in themanner described in this Example 49, or a similar manner.

Example 50 Post-DNA Gel Staining

Agarose gels (1% agarose) were prepared following a standard protocol(J. Roskams et al., Lab Ref A Handbook of Recipes, Reagents, and OtherReference Tools for Use at the Bench, Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y., 2002). Serial two-fold dilutions of 1kb Plus DNA Latter from Invitrogen Co. (Carlsbad, Calif.) were made andthe resulting DNA samples were loaded onto an agarose gel in four lanesfrom left to right with a loading of 200 ng, 100 ng, 50 ng, and 25 ngper lane, respectively. The DNA samples were electrophoreticallyseparated in 1×TBE buffer using a standard protocol. The nucleic acidsample was loaded onto the gel from in four columns from left to rightwith a loading of 200 ng, 100 ng, 50 ng, and 25 ng per lane,respectively, and electrophoretically separated in 1×TBE buffer using astandard protocol. A stock solution of a Dye No. 20 of Table 1 in DMF atabout 12 mM concentration was prepared. The stock solution of dye wasdiluted using an appropriate aqueous solvent to provide a stainingsolution with an appropriate effective working concentration of the dye,which for Dye No. 20 was about 3.6 μM in TBE solvent. The agarose gelwas submerged in the staining solution for approximately 30 minutes tostain the gel. The resulting gel was then viewed using a UVtransilluminator with 254 nm excitation. Photographs of the fluorescentimages of the illuminated gels were taken with a SYBR filter andPolaroid 667 black-and-white film.

A similar procedure was followed to prepare post gel staining solutionsof Dye No. 29 of Table 1, except that Dye No. 29 was used in place ofDye No. 20 and at various conditions, as now described. Briefly, a stocksolution of 12 mM Dye No. 29 in DMF was diluted 3,300 times separatelywith water comprising 0.1 M NaCl, with 1×TBE buffer, and with 1×TBEbuffer comprising 0.1 M NaCl, thereby providing, respectively, threeseparate staining solutions, as follows: 3.6 μM in water with 0.1 MNaCl, 3.6 μM in 1×TBE, and 3.6 μM in 1×TBE with 0.1 M NaCl. IdenticalDNA samples were prepared, loaded onto three separate agarose gels, andseparated via standard gel electrophoreses, in the manner describedabove. Each of the three separate gels was then stained by immersion ina different one of the resulting gels for about 30 minutes. Theresulting stained gels were then viewed using either a UVtransilluminator or a Dark Reader transilluminator and the resultingimages were photographed using a SYBR filter and Polaroid 667black-and-white film. Photographic results obtained using the first,second and third of these staining solutions are shown in FIGS. 9 (viaUV transilluminator), 11 (via Dark Reader transilluminator) and 14 (viaUV transilluminator), FIG. 14 (via UV transilluminator), and FIG. 14(via UV transilluminator), respectively. It should be noted that otherdyes described herein may be prepared and employed in the mannerdescribed in this Example 50, or a similar manner.

A similar procedure was followed using, in place of the stock solutionof Dye No. 20, a stock solution of SYBR Safe from Molecular Probes, Inc.(Eugene, Oreg.) as a 10,000× solution in DMSO for the preparation a 1×SYBR Safe staining solution in 1×TBE; and separately, a stock solutionof SYBR Green I from Molecular Probes, Inc. (Eugene, Oreg.) as a 10,000×solution in DMSO for the preparation of three separate stainingsolutions, as follows: a staining solution of 1× SYBR Green I in 1×TBE;a staining solution of 1× SYBR Green I in 1×TBE with 0.1 NaCl; and astaining solution of 1× SYBR Green I in water with NaCl. Identical DNAsamples were prepared, loaded onto four separate agarose gels, andseparated via standard gel electrophoreses, in the manner describedabove. Each of the four separate gels was then stained by immersion in adifferent one of the resulting gels for about 30 minutes. The resultingstained gels were then viewed using a UV transilluminator with 254 nmexcitation and the resulting images were photographed using a SYBRfilter and Polaroid 667 black-and-white film.

Photographs of various of the illuminated gels described above are shownin FIGS. 8, 9, 11 and 14, as mentioned above in the brief description ofthese figures, and as described previously herein.

Example 51 Excitation and Emission Spectra of Dye No. 29 of Table 1 inthe Presence of dsDNA

Salmon sperm dsDNA from Sigma (St. Louis, Mo.) was dissolved in pH 7 PBSbuffer to provide a 10 mM/mL DNA solution. An aliquot of a stocksolution of 12 mM Dye No. 29 of Table 1 in DMF was added to the DNAsolution to provide a DNA-dye solution with a 0.1 μM dye concentration.The DNA-dye solution was incubated at room temperature for 1 hour. Theexcitation and emission spectra of the resulting solution with Dye No.29 at 0.1 μM were measured using a Jasco fluorescence spectrophotometerat room temperature, as graphically shown in FIG. 10. The resultsdemonstrate that Dye No. 29 can be efficiently excited by 254 nm UVlight and by a visible light with wavelength in the range from about 460nm to about 510 nm. This makes it possible to use a UV-lighttransilluminator or a visible-light transluminator, such as a DarkReader or a 488 nm laser-based gel scanner, for the reading of gels thathave been stained with the dye.

Example 52 Stability of Dye No. 35 of Table 1 and SYBR Gold in TBEBuffer

SYBR Gold 10,000× in DMSO from Molecular Probes, Inc. (Eugene, Oreg.)was diluted to 1× working concentration, as recommended by themanufacturer, in 1×TBE buffer. A stock solution of Dye No. 35 of Table 1in DMSO (12 mM) was diluted to 1.2 μM in 1×TBE buffer. The opticaldensity associated with each of the solutions was monitored at theabsorption maximum of the dye (488 nm for SYBR Gold and 500 nm for DyeNo. 35) over the course of a 24-hour period at room temperature. Agraphical representation of normalized absorbance versus time for eachof the solutions is shown in FIG. 13. As shown, the absorbanceassociated with Dye No. 35 was relatively constant over the 24-hourperiod, while the absorbance of SYBR Gold decreased by nearly 50% overthe same period. This demonstrates that Dye No. 35 is relatively morestable than SYBR Gold, as previously described herein.

Example 53 Mutagenicity of EB and Dye No. 35 of Table 1

Mutagenicity assays were carried out using a Muta-ChromoPlate test kitfrom EBPI (Brampton, Ontario, Canada). Dye No. 35 of Table 1 and EB wereassayed using three dose levels (0.25 nmole, 2.5 nmoles and 25 nmoles),respectively, for each dye, and in the absence and presence of S9extract for each dose level and each dye, respectively. A solutionwithout a dye (zero dose level) was assayed in the absence and presenceof S9 extract, as a control or negative control. S9 extract is a ratliver extract that comprises various metabolic enzymes. The testsassociated with the presence of S9 extract were undertaken to provideinformation on the potential genotoxicity of the metabolized dyes.Bacterium strain TA98, a frame shift indicator, was used for the testssince EB is a known frame shift mutagen as shown by Ames Test using thesame bacterial strain. Each single test was carried out using 36 wellsand the number of positive wells out of each 36 wells was taken as anindicator of the relative mutagenicity levels of the dye under the testconditions. The results, shown in Table 2 herein, suggest that Dye No.35 may be less mutagenic than EB. Dye No. 35 may be particularlyadvantageous relative to EB given its relatively higher sensitivity ingel staining applications and its relatively greater safety in terms oftoxicity or mutagenicity. Further, as the monomeric dye constituent inDye No. 35 is structurally similar to EB, Dye No. 35 and EB have similaror essentially the same excitation and emission spectra, such that DyeNo. 35 may be used in place of EB relatively easily, for example,without the need to change the transilluminator, such as a UVtransilluminator that is used in association with EB and may be usedwith Dye No. 35, or the filter that is used in photographingtransilluminated images.

A dimeric dye has been described herein. Such a dye may have any of anumber of desirable properties, such as relatively low backgroundfluorescence, good fluorescent signal strength, and good stability, forexample. Generally, such a dye having at most one positive charge mayhave application in the detection of the presence or absence of nucleicacid immobilized in a matrix or on a solid surface.

Useful dimeric dyes have been described herein. By way of example, adimeric dye that is suitable for detecting the presence or absence ofimmobilized nucleic acids in a gel matrix or on a solid surface has beendescribed. Useful methods for nucleic acid gel staining have also beendescribed herein. By way of example, a method of using a dye, such as amonomeric dye or a dimeric dye, for example, and a suitable salt, suchas a salt comprising an anion associated with a strong acid and a cationassociated with a strong base in a suitable amount, for post-nucleicacid gel staining, has been described. Useful methods of preparing anyof various dyes described herein and useful methods of using any ofthese dyes have also been described. Useful kits suitable fordetermining immobilized nucleic acids, which comprises a suitable dyedescribed herein, have also been described.

Various modifications, processes, as well as numerous structuresrelating to the description herein may be applicable, as will be readilyapparent to those of ordinary skill in the art, upon review of thespecification. Various references, publications, provisional andnon-provisional United States or foreign patent applications, and/orUnited States or foreign patents, have been identified herein, each ofwhich is incorporated herein in its entirety by this reference. Variousaspects and features may have been explained or described in relation tounderstandings, beliefs, theories, underlying assumptions, and/orworking or prophetic examples, although it will be understood that anysuch understanding, belief, theory, underlying assumption, and/orworking or prophetic example is not binding. Although the variousaspects and features have been described with respect to variousembodiments and examples herein, it will be understood that any of sameis not limiting with respect to the full scope of the appended claims.

1. A method of staining a sample comprising a nucleic acid immobilizedon a surface or embedded in a gel matrix, the method comprising thesteps of: a) contacting the nucleic acid with an aqueous solutioncomprising a fluorescent nucleic acid dye and a salt at a concentrationfrom about 5 mM to about 0.5 M , inclusive, wherein the salt comprisesan anion that would be sufficient as a component of a strong acid and acation that would be sufficient as a component of a strong base; b)detecting a fluorescent signal associated with said fluorescent nucleicacid dye; wherein the method produces a higher fluorescent signal than acorresponding method performed by contacting said nucleic acid with anaqueous solution lacking said salt.
 2. The method of claim 1, wherein aconcentration of the salt is about 0.05 M to about 0.2 M, inclusive. 3.The method of claim 1, wherein the strong acid has a pKa of about 2 orless and the strong base has a pKa of about 10 or more.
 4. The method ofclaim 1, wherein the salt is selected from NaCl, sodium bromide, sodiumsulfate, potassium chloride, potassium bromide, potassium sulfate,magnesium choride and tetramethylammonium chloride.
 5. The method ofclaim 1, wherein the fluorescent nucleic acid dye is a monomeric dye ora dimeric dye.
 6. The method of claim 1, wherein the dye is anasymmetric cyanine dye.
 7. The method of claim 6, wherein the dye isSYBR® Green.
 8. The method of claim 5, wherein the dye is a dimeric dye.9. The method of claim 8, wherein the dimeric dye has the formula:

wherein BRIDGE is a substantially aliphatic linker comprising from about8 to about 150 non-hydrogen atoms, inclusive, and wherein the linkercomprises no more than one positive charge; Q₁ is a fluorescent nucleicacid dye constituent; Q₂ is a fluorescent nucleic acid dye constituent;and Q₁ and Q₂ may be the same or different, wherein (i) when Q₁ and/orQ₂ is a phenanthridinium dye, at least one of Q₁ and Q₂ is aphenanthridinium dye having the structure of Formula I:

R₁ represents where BRIDGE attaches to the structure; and Ψ is an anion;or (ii) when each of Q₁ and Q₂ is an asymmetric cyanine dye, each of theQ₁ and Q₂ dye constituents has the structure of Formula II:

wherein R₁′ of Formula II is H; alkyl or alkenyl having 1 carbon to 6carbons, inclusive; a halogen; —OR₉; —SR₁₀; —NR₁₁R₁₂; —CN; —NH(C═O)R₁₃;—NHS(═O)₂R₁₄; —C(═O)NHR₁₅; or a substituent associated with minor groovebinding; or, represents where BRIDGE attaches to the structure; when R₁′of Formula II comprises at least one of R₉, R₁₀, R₁₁, R₁₂, R₁₃, R₁₄ andR₁₅, any said one of R₉, R₁₀, R₁₁, R₁₂, R₁₃, R₁₄ and R₁₅, independently,is H or alkyl having 1 carbon to 12 carbons, inclusive, optionallyincorporating 1 to 2 nitrogen(s), inclusive, or an aryl; when R₁′ ofFormula II comprises R₁₁ and R₁₂, R₁₁ and R₁₂ may in combination form a5- or 6-membered, saturated or unsaturated ring, which optionallycomprises at least one hetero atom selected from N and O; X of FormulaII is selected from O and S; n of Formula II is selected from 0, 1, and2; R₆ of Formula II is H; alkyl or alkenyl having 1 carbon to 10carbons, inclusive, optionally comprising at least one hetero atomselected from N, O, and S; a halogen; —OR₁₆; —SR₁₆; —NR_(I6)R₁₇; or asubstituted or an unsubstituted aryl, optionally comprising 1 to 3hetero atom(s), inclusive, selected from N, O, and S; or representswhere BRIDGE attaches to the structure; R₇ of Formula II is H; alkyl oralkenyl having 1 carbon to 10 carbons, inclusive, optionally comprisingan aryl and at least one hetero atom selected from N, O, and S; or asubstituted or an unsubstituted aryl optionally comprising 1 to 3 heteroatom(s), inclusive, selected from N, O, and S; or represents whereBRIDGE attaches to the structure; R₈ and R₈′ of Formula II incombination form a fused aromatic ring, which may be further substituted1 to 4 time(s), inclusive, independently, by C1-C2, inclusive, alkyl,C1-C2, inclusive, alkoxy, C1-C2, inclusive, alkylmercapto, or a halogen;each of R₁₆ and R₁₇ independently is H; alkyl having 1 carbon to 12carbons, inclusive, optionally incorporating 1 to 2 nitrogen(s) or anaryl; or R₁₆ and R₁₇ may in combination form a 5- or 6-memberedsaturated or unsaturated ring, which optionally comprises at least onehetero atom selected from N and O; only one of R₁ ′, R₆, and R₇ ofFormula II represents where BRIDGE attaches to the structure; and Ψ ofFormula II is an anion; or (iii) when either Q₁ or Q₂ is an acridinedye, at least one dye constituent of the Q₁ and Q₂ dye constituents hasthe structure of Formula III:

wherein each R₁ of Formula III is independently, is H or a C1-C2,inclusive, alkyl; one of R₂ and R₃ of Formula III represents whereBRIDGE attaches to the structure; when R₂ of Formula III representswhere BRIDGE attaches to the structure, R₃ is H or —CH₃; when R₃ ofFormula III represents where BRIDGE attaches to the structure, R₂ isselected from H, —CH₃, —NH₂, —NHCH₃, —CN, and —C(═O)NH₂; each R₆ ofFormula III independently, is H or a C1-C2, inclusive, alkyl; each R₇ ofFormula III independently, is H or a C1-C2, inclusive, alkyl; for eachpair of adjacent R₆ or R₇ and R₁, independently, R₆ or R₇ and R₁ may incombination form a 5- or 6-membered, saturated or unsaturated ring; andΨ of Formula III is an anion.